JP2006314314A - Magnetic micro-particle and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a magnetic micro-particle slightly causing non-specific adsorption and to provide a method for producing the magnetic micro-particle. <P>SOLUTION: The method for producing the magnetic micro-particle comprises the following step: (a) a step of removing polypeptides other than a fusion protein of the objective polypeptide to an anchor protein from the surface of the magnetic micro-particle in which the fusion protein is expressed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a magnetic fine particle, a production method thereof, a cell separation method, and a method for separating a target molecule.

  In recent years, various biocompatible materials have been developed for the purpose of application to a wide range of fields mainly in the medical field such as artificial organs, drug delivery systems, cell engineering devices, and biochips. Among these, since magnetic particles can be easily magnetically recovered, they are expected to be applied to protein immobilization carriers, drug carriers, cell separation techniques, and the like for biosensors and immunosensors. For this reason, many studies have been conducted on methods for producing magnetic fine particles and their applications.

  As a method for producing magnetic fine particles, for example, a liposome having a diameter of 30 nm or less in which iron ions are encapsulated is produced, and a 3-5 nm magnetite is produced inside the liposome by controlling the reaction field, or iron ions are encapsulated. A method for producing silica film magnetite particles with a diameter of 5-7 nm covered with a silica film by preparing a reverse micelle of a surfactant and adding silicon dioxide to the reaction field has been reported. In addition, for example, by adding a protein having activity against silicon dioxide to the reaction field and fusing reverse micelles enclosing magnetite particles having a silica film, the magnetite particles having a plurality of silica films are included and have a diameter of around 50 nm. A method for producing protein-containing nanosilica particles has been reported.

  On the other hand, magnetic fine particles produced in the cells of magnetic bacteria are known. The magnetic microparticles or the cells themselves can be easily separated from the solution by using a magnet, so that they are useful for the production and isolation of proteins and the recovery, search, detection and quantification of various substances. is there. These magnetic fine particles are coated with an organic film mainly composed of phospholipids called a magnetic fine particle film, and various membrane proteins exist in this film.

  For example, MagA protein, MpsA protein, Mms16 protein, and the like have been identified as membrane proteins present on the magnetic fine particle film of the AMB-1 strain (Japanese Patent Laid-Open No. 8-228878, pamphlet of International Publication No. 97/35964, JP 2002-176989 A). In addition, these membrane proteins are used as anchor proteins, and luciferase (Nakamura, T., et al., J. Biochem., 118, 23-7 (1995)), protein A (Tanaka, T., et al Anal. Chem., 72, 3518-22 (2000)), G protein-coupled receptors (Yoshino, T., et al., Appl. Environ. Microbiol., 70, 2880-5 (2004)) Methods for expressing useful proteins have been developed.

On the other hand, for example, in the applications described above, in order to improve measurement accuracy, development of magnetic fine particles having excellent dispersibility and low nonspecific adsorption is required.
JP-A-8-228782 International Publication No. 97/35964 Pamphlet JP 2002-176989 A Nakamura, T., et al., J. Biochem., 118, 23-7 (1995) Tanaka, T., et al., Anal. Chem., 72, 3518-22 (2000) Yoshino, T., et al., Appl. Environ. Microbiol., 70, 2880-5 (2004)

  It is an object of the present invention to provide a magnetic fine particle with less non-specific adsorption and a method for producing the same, and a cell separation method and a target molecule separation method using the magnetic fine particle.

1. The method for producing magnetic fine particles of the present invention comprises:
(A) a step of removing a polypeptide other than the fusion protein from the surface of the magnetic fine particle expressing the fusion protein of the target polypeptide and the anchor protein.

  In the present invention, “polypeptide” refers to a substance containing a plurality of peptide bonds, and is a concept encompassing both “peptide” and “protein”. In general, “peptide” refers to a polymer containing a polypeptide with a molecular weight of 10,000 or less, and “protein” refers to a polymer containing a polypeptide with a molecular weight of 10,000 or less.

  In the present invention, the “target polypeptide” refers to any polypeptide that is desired to be expressed on the surface of magnetic fine particles.

  Furthermore, in the present invention, “fusion protein” refers to a protein that is not found in nature, in which two or more proteins or fragments thereof are linked. A fusion protein can be produced by ligating genes encoding respective proteins or fragments thereof in-frame and expressing them recombinantly.

  Furthermore, in the present invention, the “anchor protein” is a protein that is expressed by binding a part or all of it on a cell membrane or a magnetic fine particle membrane, and serves to anchor a fusion protein containing the protein to the membrane. Have

  Here, in the method for producing magnetic fine particles of the present invention, the magnetic fine particles used in the step (a) have a film on the surface, and the step (a) includes a polypeptide other than the fusion protein, The method may include a step of removing the film, and may further include a step of (b) forming another film on the surface of the magnetic fine particles after the step (a).

  Here, in the method for producing magnetic fine particles of the present invention, before the step (a), (c) a DNA construct into which a DNA encoding the fusion protein is inserted is introduced into a bacterium, Can be further produced by producing the magnetic microparticles and isolating the magnetic microparticles from the bacteria.

In this case, the bacterium may be a magnetic bacterium. In this case, the DNA construct includes an expression vector,
The expression vector may contain a DNA fragment that can be transcriptionally controlled in magnetic bacteria as a promoter, and DNA encoding the fusion protein may be inserted downstream of the DNA fragment.

  In this case, the DNA fragment can be the DNA fragment described in any of (1) to (3) below.

(1) a DNA fragment having a base sequence represented by any one of SEQ ID NOs: 1, 2, 3 and 4;
(2) A nucleotide sequence represented by any one of SEQ ID NOs: 1, 2, 3 and 4 has a nucleotide sequence in which one or more bases are substituted, deleted or added, and has promoter activity in bacteria DNA fragments having,
(3) A DNA fragment having a base sequence having 80% or more identity with the base sequence represented by any of SEQ ID NOs: 1, 2, 3, and 4 and having promoter activity in bacteria. Any of the above DNA fragments (1) to (3) exhibits high promoter activity in magnetic bacteria.

  In the method for producing magnetic fine particles of the present invention, the anchor protein may be a magnetic bacterial particle membrane protein or a fragment thereof.

  Here, in the method for producing magnetic fine particles of the present invention, the anchor protein can be Mms13.

  Here, in the method for producing magnetic fine particles of the present invention, in the step (a), at least one selected from urea, thiourea, a surfactant, a base, and a salt is used. Other polypeptides can be removed.

  Here, in the method for producing magnetic fine particles of the present invention, in the step (b), the other film can be formed using phospholipid.

  Here, in the method for producing magnetic fine particles of the present invention, the anchor protein may have an amino acid sequence represented by SEQ ID NO: 8.

  Here, in the method for producing magnetic fine particles of the present invention, the anchor protein can have at least amino acid 1-87 in the amino acid sequence represented by SEQ ID NO: 8.

  According to the method for producing magnetic microparticles of the present invention, the method includes (a) removing a polypeptide other than the fusion protein from the surface of the magnetic microparticle expressing the fusion protein of the target polypeptide and the anchor protein. Thus, it is possible to obtain magnetic fine particles which do not contain unnecessary proteins and have little nonspecific adsorption. Thereby, magnetic fine particles with low antigenicity can be obtained. Therefore, it can be applied to a living body or a sample collected from the living body (for example, blood components and cells).

  Moreover, according to the method for producing magnetic fine particles of the present invention, the magnetic fine particles used in the step (a) have a film on the surface, and the step (a) includes a polypeptide other than the fusion protein, The method includes a step of removing the film, and after the step (a), further includes (b) a step of forming another film on the surface of the magnetic fine particles, thereby providing excellent dispersibility and non-specific adsorption. Since the amount is small, magnetic particles having low antigenicity can be obtained.

  More specifically, according to the method for producing magnetic fine particles of the present invention, by linking a structural gene (DNA) encoding the fusion protein downstream of a DNA fragment that functions as a promoter and introducing it into bacteria, The fusion protein can be expressed efficiently and in large quantities in the magnetic microparticle membrane, cell membrane or cytoplasm of magnetic bacteria.

The magnetic fine particles of the present invention are
It can be obtained by removing the polypeptide other than the fusion protein from the surface of the magnetic fine particle expressing the fusion protein of the target polypeptide and the anchor protein.

The magnetic fine particles of the present invention are
A step of removing the polypeptide other than the fusion protein and the film from the surface of the magnetic fine particle expressing the fusion protein and film of the target polypeptide and anchor protein, and forming another film on the surface of the magnetic fine particle It is obtained by the process to do.

  Here, in the magnetic fine particles of the present invention, the anchor protein can have an amino acid sequence represented by any one of SEQ ID NOs: 5 to 8.

  Here, the magnetic fine particles of the present invention may have at least amino acids 1-87 in the amino acid sequence represented by SEQ ID NO: 8.

  Here, in the magnetic fine particles of the present invention, the target polypeptide may be an antibody.

  The method for separating a target molecule of the present invention comprises a step of bringing the magnetic fine particles of the present invention into contact with a target molecule that specifically binds to the target polypeptide and magnetically separating the target molecule.

  In the magnetic separation method of the present invention, when the target polypeptide is an antibody in the magnetic microparticle of the present invention, the magnetic microparticle of the present invention is brought into contact with a cell that specifically binds to the antibody. Magnetic separation can be included.

1. Method for Producing Magnetic Fine Particles The method for producing magnetic particles of the present invention comprises (a) a step of removing a polypeptide other than the fusion protein from the surface of magnetic fine particles expressing a fusion protein of the target polypeptide and an anchor protein. including.

  Here, the magnetic fine particles used in the step (a) can have a film on the surface thereof. In this case, the step (a) may include a step of removing the film together with a polypeptide other than the fusion protein. After the step (a), (b) another film is formed on the surface of the magnetic fine particles. A forming step may be further included.

  Here, before the step (a), (c) introducing a DNA construct into which a DNA encoding a fusion protein is inserted into a bacterium, culturing the bacterium to produce the magnetic fine particles, The method may further comprise isolating the magnetic microparticles from bacteria.

  Hereinafter, each of the steps (a) to (c) will be described.

1.1. Step (c)
The step (c) is a step of producing magnetic fine particles (hereinafter also referred to as “raw material magnetic fine particles”) as a raw material in the method for producing magnetic fine particles of the present invention. The raw magnetic fine particles used in the method for producing magnetic fine particles of the present invention can be obtained by the step (c).

  In the present invention, as the raw magnetic particles, those produced by bacteria (preferably magnetic bacteria) can be used. More specifically, by introducing a DNA construct into which a DNA encoding a fusion protein of a target polypeptide and an anchor protein is inserted into a bacterium (preferably a magnetic bacterium), and culturing the bacterium, the raw magnetic particles are obtained. The raw magnetic particles can be obtained by producing and isolating the raw magnetic particles from the bacteria.

1.1.1. Magnetic bacteria Magnetic bacteria are bacteria that have the ability to accumulate magnetic particles in the body. In the present invention, magnetic bacteria can be used to produce magnetic fine particles by culturing.

  Examples of magnetic bacteria that can be used in the present invention include microorganisms of the Magnetospirillum species (for example, Magnetospirillum magneticum AMB-1 (FERM BP-5458), MS-1 (IFO 15272, ATCC31632, DSM3856), SR-1 (IFO 15272). DSM 6361), and microorganisms of the species Desulfovibrio (for example, Desulfovibrio sp. RS-1 (FERM P-13283)), etc. In the cells of magnetic bacteria, fine particles composed of magnetite having a particle size of about 50 to 150 nm are contained. Produced and referred to as magnetic microparticles.

1.1.2. DNA construct In the method for producing magnetic fine particles of the present invention, the DNA construct may contain an expression vector. In this case, the expression vector can include a DNA fragment that can be transcriptionally controlled in magnetic bacteria as a promoter, and DNA encoding the fusion protein can be inserted downstream of the DNA fragment (see, for example, FIG. 12). ). Such a DNA fragment is not particularly limited as long as transcription can be controlled in a magnetic bacterium. For example, the DNA fragment described in any of (1) to (3) below is preferable.

(1) a DNA fragment having a base sequence represented by any one of SEQ ID NOs: 1, 2, 3 and 4;
(2) A nucleotide sequence represented by any one of SEQ ID NOs: 1, 2, 3 and 4 has a nucleotide sequence in which one or more bases are substituted, deleted or added, and has promoter activity in bacteria DNA fragments having,
(3) A DNA fragment having a base sequence having 80% or more identity with the base sequence represented by any of SEQ ID NOs: 1, 2, 3, and 4 and having promoter activity in bacteria.

  Any of the above DNA fragments (1) to (3) exhibits high promoter activity in magnetic bacteria.

  As described in Japanese Patent Application No. 2004-263832, DNA fragments (1) to (3) are obtained by isolating a cell membrane fraction from a magnetic bacterium, and a protein having a high expression amount among proteins contained in this fraction. And the promoter activity in the upstream region of the DNA encoding this protein was identified.

  Further, DNA fragments (1) to (3) can be obtained by direct cloning from the genome of a magnetic bacterium using a conventional method such as a PCR method based on the sequence information disclosed in the present specification. . Alternatively, the DNA fragment of the present invention may be chemically synthesized.

1.1.2 A. DNA fragment (1)
The nucleotide sequences represented by SEQ ID NOs: 1, 2, 3 and 4 (DNA fragment (1)) are proteins Msp1 (mm10) and Msp2 (mm24) expressed on the cell membrane or magnetic particle membrane of magnetic bacteria, respectively. , Msp3 (mm1) and Mms16 (mms16) corresponding to the sequence of the putative promoter region of DNA. The base sequences of the promoter regions (Pmsp1, Pmsp2, Pmsp3, and Pmms16) of these genes are shown in SEQ ID NOs: 1, 2, 3, and 4, respectively.

  All of these DNA fragments were found to exhibit high promoter activity in magnetic bacteria. FIG. 10 shows the sequences of positions −35 and −10 of these promoter regions and the sequence of the ribosome binding site.

  The base sequences represented by SEQ ID NOs: 1, 2, 3 and 4 each represent a sequence having a length of 500 bases upstream from the start codon position, and 5 ′ of these base sequences as long as it has a desired promoter activity. The side sequence may be even shorter. Preferably, the DNA fragment (1) is 300 bases, preferably 200 bases, more preferably 170 bases, more preferably 150 bases on the 3 ′ end side in the sequence shown in SEQ ID NO: 1, 2, 3 or 4. It has the arrangement | sequence.

1.1.2B. DNA fragment (2)
The DNA fragment (2) has one or more bases substituted, deleted or added in the base sequence represented by SEQ ID NO: 1, 2, 3 or 4 and has promoter activity in bacteria.

  Based on the nucleotide sequence of the natural gene disclosed in the present specification, a method for modifying a gene by deleting a part of the nucleotide of the sequence or adding or substituting another nucleotide is known in the art. Well known in the field. Examples of such methods include restriction enzyme digestion, exonuclease digestion, insertion of other DNA fragments, partial specific mutagenesis, PCR using a mutagenesis primer, and the like.

1.1.2C. DNA fragment (3)
The DNA fragment (3) has a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 1, 2, 3 or 4, and has promoter activity in bacteria. Preferably, the DNA fragment (3) has 85% or more identity, more preferably 90% or more identity, and further preferably 95% identity with the base sequence represented by SEQ ID NO: 1, 2, 3 or 4. Have sex. The DNA fragment (3) may be prepared using the gene modification method described above, or may be isolated from magnetic bacteria of various species or strains.

  The identity of the base sequence can be determined using a homology search algorithm known in the art. One example of such an algorithm is BLAST, and its use is well known to those skilled in the art.

1.1.2 D. Measurement of promoter activity The promoter activity of the DNA fragment of the present invention is introduced into bacterial cells by linking a reporter gene downstream (3 ′ end side) of the DNA fragment, for example, as described in Example 2 described later. Then, it can be evaluated by measuring the expression of the reporter gene. Examples of such reporter genes include luciferase, CAT, β-galactosidase, alkaline phosphatase, GFP and the like.

1.1.2 E.E. Expression vector In another aspect, the present invention provides an expression vector comprising the promoter of the present invention (for example, any one of the above DNA fragments (1) to (3)). In the present invention, an “expression vector” is a vector that can be replicated in bacteria and can direct the expression of a fusion protein under the control of the promoter of the present invention.

  Any known vector that can replicate in bacteria can be used as the vector into which the promoter of the present invention can be incorporated. In particular, when a magnetic bacterium is used as the bacterium, for example, pRK415 described in JP-A-8-228782, pMS-T1 described in JP-A-11-285387, or a derivative thereof is preferably used. it can.

  The expression vector has a promoter of the present invention, an origin of replication, and a selection marker for facilitating selection of a bacterium introduced with the vector, and preferably a restriction for facilitating gene insertion downstream of the promoter. Has an enzyme site. Furthermore, it may have a 3 'untranslated region for enhancing and stabilizing the expression of the inserted gene.

1.1.2 F. Introduction of DNA construct In order to transform a bacterium by introducing a DNA construct into the bacterium, any method known in the art may be used, for example, conjugation transfer, microinjection, electroporation, A known method such as a gene gun can be used. Methods for cultivating transformed bacteria and recovering the expressed protein are well known in the art and are exemplified in the examples below.

1.1.3. Raw Magnetic Fine Particles A fusion protein of a target polypeptide and an anchor protein is expressed on the surface of the raw magnetic fine particles of the present invention. When the raw magnetic microparticles are produced by magnetic bacteria, the fusion protein is expressed in the magnetic microparticle film, cell membrane, or cytoplasm of the magnetic bacteria.

  Hereinafter, the fusion protein, and the target polypeptide and anchor protein constituting the fusion protein will be described.

1.1.3A. Target polypeptide In the magnetic microparticles of the present invention, the target polypeptide may be a protein, a peptide, or both, such as various enzymes, cytokines, cell growth factors, receptors, signal transduction factors, transcription factors, Examples include transport proteins.

  In addition, by expressing the target polypeptide whose function is unknown in the raw magnetic particles of the present invention, the magnetic microparticles of the present invention can be used for elucidating the function of the target polypeptide. Alternatively, for the purpose of screening for a target polypeptide having a desired function, a library such as cDNA may be used as DNA encoding the target polypeptide.

1.1.3B. Anchor protein The anchor protein should just be stably existing on a magnetic microparticle. In magnetic bacteria, various proteins (magnetic bacterial particle membrane proteins) existing on cell membranes or magnetic fine particle membranes have been identified so far, and any of them can be used as an anchor protein.

  As the anchor protein, for example, magnetic bacterial particle membrane proteins derived from magnetic bacteria of the Magnetospirillum species (for example, Mms5, Mms6, Mms7, Mms13, etc.) are preferably used. Mms5, Mms6, Mms7, and Mms13 each have an amino acid sequence represented by SEQ ID NOs: 5 to 8.

  In addition, DNA sequences encoding the amino acid sequences represented by SEQ ID NOs: 5 to 8 are all registered in the DDBJ / GENBANK (trademark) / EBI data bank, and their access numbers are AB096081 and AB096082 (Arakaki, A., et al., J. Biol. Chem., 278, 8745-50 (2003)).

  Furthermore, a fragment of a magnetic bacterial particle membrane protein that has a function of directing localization to the magnetic fine particle membrane can also be used in the present invention to produce a fusion protein with the target polypeptide.

1.1.3C. Fusion protein The fusion protein expressed on the surface of the magnetic microparticle of the present invention is produced by linking the target polypeptide and a gene encoding an anchor protein or a fragment thereof in-frame and recombinantly expressing them. Can do.

  A plurality of different types of fusion proteins may be expressed on the surface of one magnetic fine particle. In this case, “a plurality of different types of fusion proteins” means, for example, a case where a plurality of fusion proteins contain different types of target polypeptides, a case where a plurality of fusion proteins contain different types of anchor proteins, or Can be both.

  In the present invention, Mms13 that can be used as an anchor protein is firmly bound to the magnetic fine particles, and therefore hardly peels from the magnetic fine particles when removing a polypeptide other than the fusion protein in the step (b) described later. Thus, in the present invention, Mms13 is particularly suitable for efficiently anchoring the fusion protein to the magnetic fine particles.

  The amino acid sequence of Mms13 is known (Arakaki, A., et al., J. Biol. Chem., 278, 8745-50 (2003)). Mms13 is a membrane twice-penetrating protein consisting of 124 amino acids, and the N and C terminus are thought to be localized on the cell membrane surface. Therefore, the fusion protein produced using the entire region of Mms13 can be anchored onto the cell membrane or magnetic particle membrane. Mms13 includes a C-terminal region, that is, a region that is not a transmembrane site (amino acids 88 to 124 of the amino acid sequence shown in SEQ ID NO: 8). This region may not exist. That is, the anchor protein should just have at least amino acids 1-87 among the amino acid sequences shown by sequence number 8.

  Accordingly, in one preferred embodiment of the present invention, a fusion protein comprising at least a protein having amino acids 1-87 of the amino acid sequence shown in SEQ ID NO: 8 is provided.

  When a magnetic bacterium is transformed by incorporating a gene encoding a fusion protein into the expression vector of the present invention, and the resulting transformant is cultured under appropriate conditions, magnetic particles are produced in the cells and coated with the magnetic particles. The fusion protein is expressed in the form of anchoring to the magnetic fine particle film. After destroying or lysing the cells of the magnetic bacteria, the magnetic fine particles are collected using magnetism, whereby a fusion protein of the target polypeptide and the anchor protein can be easily obtained.

  Methods for isolating magnetic microparticles from magnetic bacteria are well known in the art and are exemplified in the examples below. Isolation of the fusion protein in the cytoplasm and cell membrane of magnetic bacteria can be performed by disrupting or lysing the cells and then fractionating them by centrifugation, and the method is well known in the art.

  In the magnetic fine particles of the present invention, the fusion protein is expressed in a form in which the target polypeptide is anchored to the magnetic fine particle film, as shown in FIG. For this reason, it is possible for the polypeptide of interest to assume its natural conformation or a conformation close thereto. Therefore, it is considered that the target polypeptide contained in the magnetic fine particles of the present invention retains its various functions such as enzyme activity and ligand binding characteristics.

1.2. Step (a)
As described above, the step (a) is a step of removing the polypeptide other than the fusion protein from the surface of the magnetic fine particles (raw material magnetic fine particles) in which the fusion protein of the target polypeptide and the anchor protein is expressed.

  As described above, the raw magnetic particles can be obtained by the above-described step (c). Further, as described above, the fusion protein of the target polypeptide and the anchor protein is expressed on the surface of the raw magnetic particles. Since the anchor protein is firmly bound to the surface of the magnetic fine particles, the polypeptide other than the fusion protein is separated from the surface of the raw magnetic fine particles in the step (a) while holding the fusion protein on the surface of the magnetic fine particles. Can be selectively removed. Thereby, magnetic fine particles with less nonspecific adsorption can be obtained.

  In the step (a), the detergent used for removing the polypeptide other than the fusion protein from the surface of the raw magnetic fine particles can be appropriately selected according to the ionic strength and hydrophobicity of the anchor protein. For example, at least one selected from urea, thiourea, a surfactant, a base, and a salt can be used as the cleaning agent.

  The surfactant is not particularly limited, and a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant can be used.

  Examples of the anionic surfactant include fatty acid series (for example, fatty acid sodium, fatty acid potassium, alpha sulfo fatty acid ester sodium), linear alkylbenzene series (for example, linear alkylbenzene sulfonate sodium), higher alcohol series (for example, Examples thereof include sodium alkyl sulfate ester, sodium alkyl ether sulfate ester), alpha olefin type (for example, sodium alpha olefin sulfonate), and normal paraffin type (for example, sodium alkyl sulfonate).

  Nonionic surfactants include, for example, fatty acid (for example, sucrose fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide), higher alcohol (for example, polyoxyethylene alkyl ether), alkylphenol System (for example, polyoxyethylene alkylphenyl ether).

  Examples of the cationic surfactant include quaternary ammonium salt systems (for example, alkyltrimethylammonium salt and dialkyldimethylammonium salt).

  More specifically, examples of the surfactant include sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium (CTAB), n-octyl-β-D-glucoside (OG), and the like.

  Examples of the base include sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide and the like. Examples of the salt include sodium chloride, potassium chloride, calcium chloride and the like.

  In addition, when a film (lipid film) is formed on the surface of the raw magnetic particles used in the step (a) (that is, when the raw magnetic particles are generated by magnetic bacteria), in the step (a), The film can be removed together with the polypeptide other than the fusion protein from the surface of the raw magnetic fine particles.

  In particular, when the raw magnetic fine particles used in the step (a) are those produced by magnetic bacteria, a film (lipid bilayer) and a membrane protein are usually present on the surface of the magnetic fine particles. Such raw magnetic fine particles are washed with the above-described cleaning agent in step (a) to remove polypeptides (membrane proteins) and membranes other than the fusion protein from the surface of the magnetic fine particles. Can do.

  In particular, when the anchor protein is Mms13, the fusion protein is held on the surface of the magnetic fine particles by washing the magnetic fine particles using the above-described cleaning agent because the anchor protein is very firmly bound to the surface of the magnetic fine particles. As such, polypeptides other than the fusion protein can be selectively removed from the surface of the magnetic fine particles.

1.3. Step (b)
Step (b) is, as described above, (b) a step of forming another film on the surface of the magnetic fine particles.

  In step (b), the other membrane can be, for example, a lipid membrane. In this case, another membrane can be formed using, for example, phospholipids or surfactants.

  Although it does not specifically limit as phospholipid, For example, phosphatidylcholine (PG), phosphatidylethanolamine (PE), cardiolipin, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid Can be used.

  In the step of forming a membrane, in addition to phospholipid, for example, a membrane can be formed using a block copolymer imitating lipid. As this block copolymer, it is the composite_body | complex which combined the hydrophilic polymer and the hydrophobic polymer, and what imitated the lipid membrane etc. can be illustrated.

  In the method for producing magnetic fine particles of the present invention, after the step (a), the method further includes step (b) (step of forming another film on the surface of the magnetic fine particles). It is possible to obtain magnetic microparticles with less attractive adsorption.

  In particular, when the raw magnetic fine particles are those produced by magnetic bacteria, in the step (b) after the step (a), the other film can be easily formed on the surface of the magnetic fine particles.

1.4. Effects According to the method for producing magnetic microparticles of the present invention, the method includes (a) removing a polypeptide other than the fusion protein from the surface of the magnetic microparticle expressing the fusion protein of the target polypeptide and the anchor protein. As a result, it is possible to obtain magnetic fine particles which do not contain unnecessary proteins and have little nonspecific adsorption. For this reason, measurement sensitivity can be improved. Moreover, since there are few nonspecific adsorption | suctions, a magnetic fine particle with low antigenicity can be obtained. For this reason, since the magnetic fine particles obtained by the method for producing magnetic fine particles of the present invention are highly usable in vivo, for example, they can be used as protein-immobilized carriers or drug carriers for biosensors and immunosensors. Yes, it can be suitably used in cell separation technology and the like.

  Further, in the method for producing magnetic fine particles of the present invention, the magnetic fine particles used in the step (a) have a film on the surface, and the step (a) includes the film together with a polypeptide other than the fusion protein. Including the step of removing, and further including the step (b) (step of forming another film on the surface of the magnetic fine particles) after the step (a), thereby providing excellent dispersibility and less non-specific adsorption. Magnetic fine particles having low antigenicity can be obtained. Further, when the magnetic fine particles are produced by magnetic bacteria, in the step (a), for example, when extracting the magnetic fine particles from the magnetic bacteria by the step of removing the polypeptide other than the fusion protein and the film, Since the outer membrane fraction (for example, membrane components and proteins) that can be mixed into the film on the surface of the magnetic particles can be removed together, magnetic particles with lower antigenicity can be obtained, and the above step (b) In this case, another film can be easily formed on the surface of the magnetic fine particles.

2. Magnetic fine particles The magnetic fine particles of the present invention can be obtained by the above-described method for producing magnetic fine particles of the present invention. That is, the magnetic fine particles of the present invention can be obtained by, for example, a step of removing a polypeptide other than the fusion protein from the surface of the magnetic fine particles expressing the fusion protein of the target polypeptide and the anchor protein. The magnetic fine particles of the present invention include, for example, a step of removing a polypeptide other than the fusion protein and the film from the surface of the magnetic fine particles expressing the fusion protein and film of the target polypeptide and anchor protein, and the magnetic fine particles. Can be obtained by a process of forming another film on the surface.

  The fusion protein constituting the magnetic fine particles of the present invention, and the target polypeptide and anchor protein constituting the fusion protein are as described above. For example, the fusion protein constituting the magnetic microparticle of the present invention can include at least a protein having amino acids 1 to 87 of the amino acid sequence represented by SEQ ID NO: 8.

3. Applications Using the magnetic microparticles of the present invention, functional analysis of target polypeptides, ligand identification and quantification, agonist / antagonist screening, cell separation, etc. can be easily performed. Hereinafter, specific examples of applications will be described.

3.1. Separation of Target Molecules The magnetic fine particles of the present invention can be used in a method for separating target molecules. In this case, for example, the target molecule can be isolated by bringing the magnetic fine particle of the present invention into contact with a target molecule that specifically binds to the target polypeptide and magnetically separating the target molecule. The target molecule is not particularly limited, and may be a polypeptide such as a protein or a peptide, or may be a low molecular compound that can be used as an active ingredient of a pharmaceutical, for example.

  For example, a substance (target molecule) that binds to a target polypeptide is detected in a sample by contacting the magnetic fine particle of the present invention with a sample and measuring the presence or amount of the substance bound to the magnetic fine particle. Can do. In particular, this method is useful for assaying a ligand of a polypeptide of interest. This method has an advantage that the substance (target molecule) bound to the target polypeptide on the magnetic fine particle can be easily separated from the unbound substance by collecting the magnetic fine particle using a magnet. Furthermore, the magnetic fine particles of the present invention are also useful for use in screening for useful substances. That is, a substance (target molecule) capable of binding to the target polypeptide is determined by contacting the magnetic fine particles of the present invention with a solution containing the test substance and determining whether or not the test substance binds to the magnetic fine particles. Can be identified. This method is useful for screening for agonists / antagonists of the polypeptide of interest. This method is also useful for identifying the ligand and analyzing the function of the target polypeptide when the ligand of the target polypeptide is unknown. Moreover, although the method of the present invention is predicted to be a polypeptide having a specific function from the sequence information, the corresponding polypeptide has not been isolated or its presence has not been confirmed. Therefore, the present invention is particularly useful for analyzing the actual function of a protein whose function is estimated from genomic sequence information or cDNA sequence information.

  Furthermore, using the magnetic fine particles of the present invention, only a substance (target molecule) that binds to the target polypeptide can be isolated from a sample containing a plurality of test substances. For example, a substance (target molecule) bound to a target polypeptide on the magnetic fine particles can be easily separated by bringing the magnetic fine particles into contact with a sample containing a plurality of test substances and collecting the magnetic fine particles using a magnet. it can. By isolating and analyzing these substances (target molecules), information useful for elucidating the function of the target polypeptide can be obtained. Also, agonist / antagonist screening can be easily performed using this method.

3.2. Cell separation When the target polypeptide bound to the fusion protein in the magnetic microparticle of the present invention is an antibody, the magnetic microparticle of the present invention is brought into contact with a cell that specifically binds to the antibody, and the cell is magnetically separated. Can do. According to this method, for example, only cells having a specific antibody can be magnetically separated from cells collected from tissues or organs of animals or plants.

4). EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

4.1. Example 1 (Identification of proteins expressed in large quantities in magnetic bacteria)
The magnetic bacterium Magnetospirillum magneticum AMB-1 (ATCC 700264) was cultured using MSGM medium (pH 6.75) (Yang, CD et al., Enzyme Microb. Technol., 29, 13-19 (2001)). The cells in the stationary culture period were collected, washed with HEPES, and then crushed by passing 3 times through a French press. Next, magnetic fine particles are separated from the French press crushed sample using a magnet, the remaining sample is centrifuged at 10000 G, 4 ° C. for 10 minutes, and the supernatant is further centrifuged at 160000 G at 4 ° C. for 1 hour. did. The precipitate was resuspended in HEPES buffer and ultracentrifuged at 160000 G for 1 hour at 4 ° C. to collect the precipitate. This was resuspended in PBS buffer to obtain a cell membrane fraction.

This cell membrane fraction was separated by 2D gel electrophoresis and transferred to a PVDF membrane by electroblotting. The result of staining the membrane with Coomassie brilliant blue is shown in FIG. A dark protein spot that is thought to have a high expression level is cut out with a scalpel and the protein sample is subjected to Edman degradation using Shimadzu amino acid sequencing system PPSQ-1 (manufactured by Shimadzu Corporation). The N-terminal amino acid sequence was determined from the genome information of the AMB-1 strain, and Msp1, Msp2 and Msp3 were identified as proteins expressed in large amounts on the magnetic bacterial membrane ( 1 and 2, spots a and b represent Msp1, spot c represents Msp2, and spot e represents Msp3.

4.2. Example 2 (Measurement of promoter activity)
About the highly expressed protein identified in Example 1, the sequence information of the genomic DNA of AMB-1 (“Complete Genome Sequence of the Facultative Anaerobic Magnetotactic Bacterium Magnetospirillum sp. Strain AMB-1”, Tadashi Matsunaga, Yoshiko Okamura, Yorikane Fukuda, Based on Aris Tri Wahyudi, Yaeko Murase, Haruko Takeyama, DNA research, 12, 157-166 (2005)), the upstream sequence of the coding sequence of Msp1, Msp2, and Msp3 was examined and the consensus sequence of bacterial promoters was compared. Thus, the promoter region was searched (in the above paper, the sequence of the promoter Pmms24 is also published). The nucleotide sequences of DNAs containing these promoter regions are shown in SEQ ID NOs: 1, 2, and 3, respectively. In addition, FIG. 3 shows the sequences near the −35 and −10 positions of these promoter regions and the sequence of the ribosome binding site. In this way, the promoter regions of the promoters Pmsp1, Pmsp2 and Pmsp3 were identified.

  Next, these promoter region DNAs were amplified from the AMB-1 genomic DNA by PCR, and inserted into a vector pUMGLC (see FIG. 4) containing a gene encoding luciferase as a reporter and capable of replication in both E. coli and magnetic bacteria. Thus, a DNA construct for expression was constructed. For comparison, the promoter PmagA (“ron-regulated expression and membrane localization of the magA protein in Magnetospirillum sp. Strain AMB-1”. C. Nakamura, T. Kikuchi, JG Burgess, T. Matsunaga. J. Biochem (Tokyo 1995 Jul; 118 (1): 23-7.), Promoter Plac, and Pmms16 (sequence represented by SEQ ID NO: 4) were similarly introduced into the expression vector. As a negative control, a vector without a promoter was used.

  The expression DNA construct thus prepared was introduced into Magnetospirillum magneticum AMB-1 by electroporation. The obtained transformant was cultured in MSGM medium containing ampicillin (5 μg / ml) (Matsunaga, T. et al., Appl. Microbiol. Biotechnol. 35, 651-655 (1991)). The cells were collected, washed with HEPES buffer, and resuspended in PBS buffer. A luciferase luminescent substrate (Promega) containing a cell lysate was added to the cell suspension, and the luminescence intensity after 1 minute was measured. The result is shown in FIG.

  As is clear from FIG. 5, when Pmsp1 and Pmsp3 were used as promoters, luciferase activity higher than that of the known promoters PmagA and Pmms16 was obtained, and in particular, when Pmsp3 was used, the highest activity was obtained.

4.3. Example 3 (Expression of fusion protein of target protein and anchor protein)
In order to efficiently localize the functional protein on the magnetic fine particle film, an anchor protein was selected. As shown in FIG. 6, a DNA construct was constructed so that a fusion protein of an anchor protein (MagA, Mms16, or Mms13) and a target polypeptide (luciferase) was expressed under the control of the promoter Pmms16. These DNA constructs were introduced into magnetic bacteria in the same manner as in Example 2, and the bacteria were cultured. The bacterial cells were collected, passed through a French press three times to crush the bacterial cells, and magnetic fine particles were isolated using a magnet. The obtained magnetic fine particles were washed with a HEPES buffer, suspended in a PBS buffer, and a luciferase luminescent substrate (Promega) was added to measure the luminescence intensity. Further, in the same manner as in the method used in Example 1, cell debris, cytoplasm, and cell membrane fractions were prepared, and the luciferase activity of each fraction was measured. The result is shown in FIG.

  As is apparent from FIG. 7, when Mms13 is used as the anchor protein, the amount of luciferase (target polypeptide) expressed in the cell is the largest, and the anchoring efficiency on the magnetic particle film is the anchor protein. As compared with the case where MagA and Mms16 were used, it was shown that it was remarkably high. This is probably because Mms13 is a small protein that penetrates the membrane twice (see FIG. 9), and thus is stably anchored to the magnetic fine particle film. Furthermore, instead of the promoter Pmms16, expression of a fusion protein of Mms13 and luciferase was evaluated under the control of the promoter Pmsp1 or Pmsp3. When the promoter Pmsp1 or Pmsp3 was used, the expression was 3 as compared with the case where the promoter Pmms16 was used. Double expression levels were obtained (see FIG. 8).

4.4. Example 4 (Stability of fusion protein of target polypeptide (luciferase) and anchor protein (Mms13) on magnetic fine particles)
In this example, the stability of a fusion protein of a target polypeptide (luciferase) and an anchor protein (Mms13) on magnetic particles was evaluated.

  An expression DNA construct (Japanese Patent Application No. 2004) constructed so as to express a fusion protein of Mms13 (anchor protein) and luciferase (target polypeptide) under the control of the promoter Pmms16 in the same manner as in Example 3. -263832) was introduced into the magnetic bacterium Magnetospirillum magneticum AMB-1 (ATCC 700264). Magnetic bacteria were cultured using MSGM medium (pH 6.75) (Yang, CD et al., Enzyme Microb. Technol. 29, 13-19 (2001)). Next, the cells in the stationary culture period were collected, washed with HEPES buffer, and then crushed by passing three times through a French press. Next, magnetic fine particles are separated from a French press crushed sample using a magnet, suspended in a HEPES buffer solution, dispersed by ultrasonic waves, and again subjected to magnetic separation using a magnet 10 times. The obtained particles were used as refined magnetic fine particles. Next, in order to evaluate the stability of the fusion protein of Mms13 (anchor protein) and luciferase (target polypeptide) by washing, the surface of the magnetic microparticles was subjected to magnetic separation and resuspension by pipetting five times. The amount of the fusion protein present was evaluated by Western blot analysis, and the enzyme activity of the magnetic microparticles was evaluated by adding a luciferase luminescent substrate (Promega).

  FIG. 10 (a) shows the result of Western blot analysis for the protein removed from the magnetic fine particles in which the fusion protein is expressed. FIG. 10 (b) shows the magnetic fine particles in which the fusion protein is expressed after washing and magnetic separation. The luminescence intensity of luciferase is shown. In Western blot analysis, the protein removed from the magnetic microparticles was analyzed. In FIG. 10A, 2.5 mg of magnetic fine particles were used, and in FIG. 10B, 20 μg of magnetic fine particles were used.

  As a result, it was shown that the fusion protein was stably present and the enzyme activity was also stably maintained after 5 washes (see FIGS. 10 (a) and 10 (b)).

  Furthermore, a urea solution (7M urea aqueous solution, 2M thiourea aqueous solution, 4% CHAPS, 40 mM Tris base) is applied to the magnetic fine particles in which the fusion protein of Mms13 (anchor protein) and luciferase (target polypeptide) after purification is expressed. Was added to remove the polypeptide and membrane (lipid bilayer membrane) other than the fusion protein (urea treatment). Moreover, the magnetic fine particles before and after the urea treatment were treated with a boiled 1% SDS solution. Each protein fraction in the 1% SDS solution used for this treatment was analyzed by SDS-PAGE and Western blot.

  FIG. 11 (a) shows the analysis results by SDS-PAGE, and FIG. 11 (b) shows the analysis results by Western blot using an anti-luciferase antibody. 11 (a) and 11 (b), (1) shows the analysis result of the film protein of the magnetic fine particles before the urea treatment, and (2) shows the analysis result of the film protein of the magnetic fine particles after the urea treatment.

  As a result, a large amount of particle membrane protein (polypeptide other than the fusion protein) was removed by the urea treatment. On the other hand, it was shown that the fusion protein of Mms13 (anchor protein) and luciferase (target polypeptide) was stably present on the surface of the magnetic fine particle even after the urea treatment (FIG. 11 (a) and FIG. 11 (b)).

4.5. Example 5 (Treatment of magnetic fine particles expressing ZZ domain of protein A)
In this example, first, magnetic particles in which a fusion protein of Mms13 (anchor protein) and the ZZ domain (target polypeptide) of protein A is expressed are prepared, and then a surfactant is applied to the magnetic particles. The membrane was formed on the surface of the magnetic fine particles by removing excess protein by adding a lipid and further adding lipid. Each step in the above method is shown in FIG.

  In this example, magnetic fine particles in which a fusion protein of Mms13 and ZZ domain is expressed are prepared, and then a magnetic protein membrane protein (fusion protein) is used with each surfactant (SDS, CTAB, or OG). And the lipid bilayer were removed, and a film was formed on the surface of the magnetic fine particles using phosphatidylcholine (PC) (see FIG. 12).

First, the pUM13ZZ plasmid was constructed. The ZZ domain is the IgG binding domain of protein A. In this example, a vector capable of expressing the ZZ domain in the AMB-1 strain was constructed. PMC18 (Amp ^r ) consisting of pMS-T1 and pUC18 described in JP-A-11-285387 was digested with SspI. Further, the promoter PMms16 of Magnetospirillum magneticum AMB-1 was used as the promoter, and Mms13 was used as the anchor protein. Promoter PMms16 and Mms13 genes were obtained from AMB-1 genome by PCR based on known sequence information. The gene encoding the ZZ domain was obtained from pEZZ18 from Amersham Biosciences. These PCR products are introduced into the above-described plasmid, and the promoter PMms16 sequence and downstream, the Mms13 coding sequence (anchoring protein coding sequence) and the ZZ domain coding sequence (the coding sequence of the target polypeptide) become in-frame. Thus, a DNA construct (plasmid pUM13ZZ) in which Mms13 (anchor protein) -ZZ domain (target polypeptide) was expressed was constructed.

  Next, in the same manner as in Example 3, a DNA construct constructed to express a fusion protein of Mms13 (anchor protein) and ZZ domain (target polypeptide) of protein A under the control of the promoter Pmms16 is prepared. The DNA construct was introduced into a magnetic bacterium.

Next, the magnetic bacteria were cultured in the same manner as in Example 1, and magnetic fine particles were extracted from the magnetic bacteria. The magnetic fine particles washed 10 times with HEPES buffer were washed 4 times with HEPES buffer containing 1M NaCl. Next, 16.7 mM dissolved in 10 mM HEPES buffer containing 1 mM SDS, 0.1 mM EDTA and 500 mM NaCl dissolved in 10 mM TES containing 100 mM NaCl and 0.1 mM CaCl ₂ . 1.5 ml of 1.6 mM CTAB dissolved in OG or 10 mM HEPES buffer was added and allowed to react for 2.5 hours. Thereafter, it was washed three times with 10 mM TES containing 100 mM NaCl and 0.1 mM CaCl ₂ . Next, 1 ml of 1 mg / ml phosphatidylcholine (PC) dissolved in 20 mM TES containing 15 mM NaCl is added to the magnetic fine particles treated with the above surfactant and reacted for 2 hours. A film was formed on the surface of the magnetic fine particles. Next, the magnetic fine particles of Example 5 having a film formed on the surface were obtained by washing 5 times with 20 mM TES containing 15 mM NaCl.

4.6. Example 6 (Evaluation of ZZ domain activity of magnetic fine particles of Example 5)
In this example, in order to evaluate the activity of the ZZ domain (target polypeptide) on the magnetic fine particles of Example 5 (magnetic fine particles having a film formed on the surface by addition of lipids after treatment with each surfactant). The alkaline phosphatase-labeled rabbit-derived anti-goat antibody (25 μg / ml) was added to the magnetic fine particles of Example 5 and incubated at room temperature for 1 hour.

  Next, after washing with PBS three times, Lumiphos 530 was added and reacted for 1 minute, and the luminescence intensity was measured with a luminometer, and this was evaluated as the activity of the ZZ domain. As a result, all of the magnetic bacterial particles obtained in Example 5 to which the alkaline phosphatase-labeled rabbit-derived anti-goat antibody was immobilized were confirmed to emit light, and the presence of the ZZ domain retaining activity on the magnetic fine particles was confirmed. (See FIG. 13 (a)).

  FIG. 13 (a) shows the evaluation results of the binding ability between the alkaline phosphatase-labeled rabbit-derived anti-goat antibody and the magnetic fine particles obtained in Example 5 (see FIG. 12). FIG. 13 (b) shows the surface activity. The analysis result by SDS-PAGE for confirming presence of the fusion protein of the anchor protein (Mms13) and the target polypeptide (ZZ domain) on the magnetic fine particle after the agent treatment is shown.

  In FIG. 13 (a) and FIG. 13 (b), “NC” is a measurement result when magnetic fine particles not expressing a ZZ domain (negative control) are used, and “PC” is the surfactant of Example 5. As a result of measurement using magnetic fine particles (positive control) before the treatment by “OG”, “OG”, “CTAB”, and “SDS” are determined by the surfactants (OG, CTAB, SDS) of Example 5, respectively. The measurement result in the case of using magnetic fine particles having a film formed on the surface after the treatment is shown.

  According to FIG. 13 (a), in Example 5, the magnetic fine particles treated with three kinds of surfactants (SDS, CTAB, OG) were confirmed to emit light, and the activity of the ZZ domain was confirmed. I was able to.

  Further, after the treatment with the surfactant, the magnetic fine particles were boiled using a 1% SDS solution to extract the film protein of the magnetic fine particles, and the results of SDS-PAGE analysis of this extract (FIG. 13). (B)), the ZZ domain activity on the magnetic fine particles obtained in Example 5 is proportional to the abundance of the fusion protein of Mms13 and ZZ domain on the magnetic fine particles. Indicated.

4.7. Example 7 (observation of magnetic fine particles of Example 5 with an electron microscope)
In this example, in the case where magnetic fine particles having a film formed on the surface were prepared after processing using SDS as a surfactant in Example 5, the magnetic fine particles in each step shown in FIG. It observed with the transmission electron microscope H700-H (Hitachi Ltd.) (refer FIG. 14 (a)-FIG.14 (d)).

  The particle surface after washing with HEPES buffer (see FIG. 14 (a)) and the particle surface after washing with HEPES buffer containing 1M NaCl (see FIG. 14 (b)) appear to be lipid bilayers. While the presence of the film was confirmed, the removal of the film from the particle surface was confirmed after the SDS treatment (see FIG. 14C). Furthermore, a film that appears to be a lipid film derived from PC was observed on the surface of the magnetic fine particles after addition of phosphatidylcholine (PC) (see FIG. 14D).

  From the above results, it was confirmed that according to each step of Example 5, after the film was once removed from the surface of the magnetic fine particles, the film was re-formed on the surface of the magnetic fine particles.

4.8. Example 8 (Separation of CD14 positive cells using magnetic fine particles of Example 5)
In this example, antibody-immobilized magnetic bacterial particles were prepared using the magnetic fine particles of Example 5. First, the magnetic fine particles of Example 5 (magnetic fine particles expressing a fusion protein of ZZ domain and Mms13) are suspended in 500 μl of rabbit-derived anti-mouse IgG antibody solution (1 mg / ml), and ultrasonically stirred every 5 minutes. For 1 hour at room temperature. Next, it was washed 5 times with 1 ml of PBS to remove unreacted antibodies, and then resuspended in 500 μl of PBS (0.1% NaN ₃ ) to prepare antibody-immobilized magnetic microparticles. Next, 10 ml of human peripheral blood was diluted with 10 mM PBS (25 ml), layered on 15 ml of HISTOPAQUE-1077 (Sigma), and centrifuged at 1800 rpm for 30 minutes. Next, the mononuclear cell layer was extracted, an appropriate amount of PBS (containing 2 mM EDTA and 0.5% BSA) was added, and centrifugal washing (1500 rpm, 6 minutes) was performed. FcR Booking Reagent (20 μl) was added to 180 μl of the pellet suspension and reacted at room temperature for 15 minutes, and the pellet suspension after centrifugal washing was made into a mononuclear cell suspension.

Next, 100 μl (˜3 × 10 ⁶ cells) of the mononuclear cell suspension was reacted with a mouse-derived anti-CD14 monoclonal antibody at 4 ° C. for 15 minutes. After centrifugal washing with PBS, 10 μg of antibody-immobilized magnetic fine particles into which an anti-mouse IgG antibody was introduced were reacted with 100 μl of the pellet suspension at 4 ° C. for 15 minutes. After centrifugal washing with PBS, 100 μl of the pellet suspension was reacted with 2 μl of FITC-labeled anti-mouse antibody at 4 ° C. for 10 minutes. After centrifugal washing with PBS, washing and magnetic recovery were repeated 7 times for the pellet suspension. Thereafter, the magnetic bacterial particle suspension containing the cells was collected and analyzed by flow cytometry to evaluate the purity of the collected cells (mononuclear cells).

  As a result of SS and FS analysis of the mononuclear cell suspension by flow cytometry, cells other than CD14 positive cells were observed before cell separation, whereas most of the other cells were removed after cell separation. (See FIG. 15 (a) and FIG. 15 (b)).

  Therefore, cell separation using the magnetic fine particles of Example 5 was performed, and the fluorescence intensity frequency of the cells after separation was observed (see FIGS. 16A to 16E). In FIG. 16 (b) to FIG. 16 (e), “PC”, “OG”, “CTAB”, and “SDS” have the same significance as in FIG. 13 (a) and FIG. 13 (b), respectively. . In FIGS. 16A to 16E, the horizontal axis indicates the emission intensity, and the vertical axis indicates the number of cells.

  As a result, it was shown that the magnetic fine particles of Example 5 can separate CD14 positive cells with a high purity of 98.0% or more (see Table 1). From this result, in Example 5, the treatment of the magnetic fine particles with a surfactant can remove proteins caused by nonspecific adsorption on the surface of the magnetic fine particles, and the implementation. In Example 5, it was considered that dispersibility was improved and reaction efficiency could be improved by forming a film (lipid film) on the surface of the magnetic fine particles.

1 shows the results of 2D gel electrophoresis of cell membrane protein fractions of magnetic bacteria in Example 1. FIG. FIG. 2 shows the protein identified on the magnetic bacterial cell membrane identified in Example 1. FIG. 3 shows the sequences near the −35 and −10 positions of the promoter region and the sequence of the ribosome binding site identified in Example 2. FIG. 4 shows the luciferase expression DNA construct used in Example 2 for measuring promoter activity. FIG. 5 shows the measurement results of promoter activity in Example 2. FIG. 6 is a diagram schematically showing the structure of a DNA construct used for expressing a fusion protein of a target polypeptide (luciferase) and an anchor protein in Example 3. FIG. 7 shows the localization evaluation of the fusion protein in the AMB-1 transformant according to the difference in anchor protein in Example 3. FIG. 8 shows the expression result of the fusion protein of Mms13 (anchor protein) and luciferase (target polypeptide) in the AMB-1 transformant according to the promoter in Example 3. FIG. 9 shows a predicted view of the localization mode of the anchor protein (Mms13) and the target polypeptide fusion protein present on the magnetic fine particles of the present invention. FIG. 10 (a) and FIG. 10 (b) show the results of stability evaluation of a fusion protein of an anchor protein (Mms13) and a target polypeptide (luciferase) on magnetic fine particles in Example 4. 11 (a) and 11 (b) show that, in Example 4, the urea treatment is effective for the surface stability of the magnetic fine particles in which the fusion protein of the anchor protein (Mms13) and the target polypeptide (luciferase) is expressed. The result evaluated about the influence is shown. FIG. 12 is a diagram schematically illustrating a method for producing magnetic fine particles in which a fusion protein of an anchor protein (Mms13) and a target polypeptide (ZZ domain) is expressed in Example 5. FIG. 13 (A) and FIG. 13 (B) show, in Example 6, the anchor protein (Mms13) and the target polypeptide (ZZ domain) present on the magnetic microparticles obtained in Example 5 (see FIG. 12). The analysis result of fusion protein of is shown. 14 (A) to 14 (D) show electron micrographs of magnetic microparticles in which a fusion protein of an anchor protein (Mms13) and a target polypeptide (ZZ domain) was measured, as measured in Example 7. . FIG. 15 shows the results of analyzing the cell separation using the antibody-immobilized magnetic fine particles obtained in Example 8 by flow cytometry. FIG. 16 shows the results of separating CD14-positive (+) cells from the mononuclear cell fraction using the antibody-immobilized magnetic fine particles obtained in Example 8 respectively.

Claims (18)

  (A) A method for producing magnetic fine particles, comprising a step of removing a polypeptide other than the fusion protein from the surface of the magnetic fine particles expressing a fusion protein of the target polypeptide and an anchor protein. In claim 1,
The magnetic fine particles used in the step (a) have a film on the surface,
The step (a) includes a step of removing the membrane together with a polypeptide other than the fusion protein,
After the step (a), the method of manufacturing a magnetic fine particle further includes the step of (b) forming another film on the surface of the magnetic fine particle. In claim 1 or 2,
Prior to the step (a), (c) a DNA construct into which DNA encoding the fusion protein is inserted is introduced into a bacterium, the bacterium is cultured to produce the magnetic fine particles, and the bacterium is A method for producing magnetic fine particles, further comprising a step of isolating the fine particles. In claim 3,
The method for producing magnetic fine particles, wherein the bacterium is a magnetic bacterium. In claim 3 or 4,
The DNA construct comprises an expression vector;
The method for producing magnetic fine particles, wherein the expression vector contains a DNA fragment that can be transcriptionally controlled in magnetic bacteria as a promoter, and a DNA encoding the fusion protein is inserted downstream of the DNA fragment. In claim 5,
The method for producing magnetic fine particles, wherein the DNA fragment is the DNA fragment according to any one of (1) to (3) below.
(1) a DNA fragment having a base sequence represented by any one of SEQ ID NOs: 1, 2, 3 and 4;
(2) A nucleotide sequence represented by any one of SEQ ID NOs: 1, 2, 3 and 4 has a nucleotide sequence in which one or more bases are substituted, deleted or added, and has promoter activity in bacteria DNA fragments having,
(3) A DNA fragment having a base sequence having 80% or more identity with the base sequence represented by any of SEQ ID NOs: 1, 2, 3, and 4 and having promoter activity in bacteria. In any one of Claims 1-6,
The method for producing magnetic fine particles, wherein the anchor protein is a magnetic bacterial particle membrane protein or a fragment thereof. In any one of Claims 1-7,
The method for producing magnetic fine particles, wherein the anchor protein is Mms13. In any one of Claims 1-8,
In the step (a), a method for producing magnetic fine particles, wherein a polypeptide other than the fusion protein is removed using at least one selected from urea, thiourea, a surfactant, a base, and a salt. In any one of Claims 1-9,
A method for producing magnetic fine particles, wherein in the step (b), the other film is formed using a phospholipid. In any one of Claims 1-10,
A method for producing magnetic fine particles, wherein the anchor protein has at least amino acids 1-87 of the amino acid sequence represented by SEQ ID NO: 8.   Magnetic fine particles obtained by a step of removing a polypeptide other than the fusion protein from the surface of magnetic fine particles expressing a fusion protein of a target polypeptide and an anchor protein. A step of removing the polypeptide other than the fusion protein and the film from the surface of the magnetic fine particle expressing the fusion protein and film of the target polypeptide and anchor protein, and forming another film on the surface of the magnetic fine particle Magnetic fine particles obtained by the process. In claim 12 or 13,
Magnetic fine particles, wherein the anchor protein has an amino acid sequence represented by SEQ ID NO: 8. In claim 12 or 13,
Magnetic fine particles, wherein the anchor protein has at least amino acids 1-87 of the amino acid sequence represented by SEQ ID NO: 8. In any one of Claims 12-15,
Magnetic fine particles, wherein the target polypeptide is an antibody.   A cell separation method comprising the steps of bringing the magnetic fine particles according to claim 16 into contact with cells that specifically bind to the antibody and magnetically separating the cells.   A method for separating a target molecule, comprising the step of bringing the magnetic fine particles according to any one of claims 12 to 15 into contact with a target molecule that specifically binds to the target polypeptide and magnetically separating the target molecule.