JP2009118802A - Method for producing biotin-labeled magnetic fine particles - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and efficiently producing a biotin-labeled magnetic fine particle. <P>SOLUTION: A fusion protein of a magnetic particle membrane protein derived from a magnetic bacterium or its fragment, and a biotin label sequence is expressed in a magnetic bacterium, and the magnetic fine particle is separated from the magnetic bacterium. The biotin label sequence is preferably formed of a specific amino acid sequence or an amino acid sequence obtained by deletion, substitution, addition and/or by the insertion of one or more amino acids in the specific amino acid sequence, and biotinylatable in the magnetic bacterium. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing biotin-labeled magnetic fine particles, and more specifically, particles produced by magnetic bacteria by expressing a fusion protein of a magnetic particle membrane protein and a biotin-labeled sequence in magnetic bacteria. The present invention relates to a method of simply labeling biotin on magnetic particles having a diameter of about 50 to 150 nm, and magnetic particles produced by the method.

In recent years, magnetic fine particles have been used as protein-immobilized carriers for biosensors and immunosensors. Since magnetic fine particles can be easily recovered by magnetism, they are expected to be applied not only to immunoassays but also to a wide range of fields such as drug carriers and cell separation techniques. One method for labeling substances in immunoassays and the like is binding between avidin or streptavidin and biotin. Since these bonds are extremely stable (dissociation constant 10 ⁻¹⁵ M), they are useful for improving detection sensitivity and performing rapid assays. For example, in the case of an immunoassay for measuring an antigen in a specimen, a biotinylated antibody bound to the antigen is reacted with labeled avidin or streptavidin, and the remaining labeling intensity is measured after washing.

  For biotinylation of an antibody or the like, various biotinylation reagents that can immobilize biotin using an amino group, carboxyl group, thiol group (sulfhydryl group or mercapto group) of a biotinylated substance are commercially available. However, since these chemical reagents bind to the protein activation site indiscriminately, there is a problem that the function of the protein is inhibited. Therefore, development of a site-specific labeling technique that does not inhibit the function of the protein has been desired.

  Protein domains recognized by biotin ligase in cells are widely conserved across biological species, and various proteins fused with this protein domain have been reported to be biotinylated in vivo (for example, Non-Patent Document 1). However, this biotinylated domain is a large polypeptide that requires a minimum of 75 amino acid residues. In order to develop a small site-specific biotinylation tag, a peptide library having a random amino acid sequence of 15 to 27 residues is constructed by mimicking the biotin peripheral sequence of various biotin enzymes and biotinylated in E. coli. The peptide sequence was searched. It has been found that a relatively short peptide named a biotin-accepting peptide is efficiently biotinylated by E. coli biotin ligase (see, for example, Non-Patent Document 2).

  On the other hand, magnetic fine particles produced in the cells of magnetic bacteria are known. Since the magnetic fine particles or the bacterial cells themselves can be easily separated from the solution by using a magnet, they are useful for protein production and isolation, and recovery, search, detection and quantification of various substances. is there. The magnetic fine particles are coated with an organic film whose main component is a phospholipid called a magnetic fine particle film, and various film proteins exist in this film (for example, see Patent Documents 1 to 3). In addition, a method for expressing a fusion protein with a target polypeptide on the surface of magnetic fine particles using this membrane protein as an anchor protein has also been reported (for example, see Patent Document 4).

JP-A-8-228782 International Publication No. 97/35964 Pamphlet JP 2002-176989 A JP 2006-314314 A Cronan, JE, The Journal of Biological Chemistry, (1990) Vol.265, pp.10327-10333 Schatz, PJ, Bio / Technology, (1993) Vol.11, pp.1138-1143

  To efficiently label nanometer-scale magnetic particles with biotin, functional groups such as amino groups must be introduced into the polymer that coats the surface of the magnetic particles, and the production process is complicated and expensive reagents are used. Therefore, commercially available biotin-labeled magnetic fine particles are extremely expensive. Further, it is not yet clear whether biotinylation occurs in the magnetic bacterium when the above-described biotin-accepting peptide is expressed on the magnetic fine particle film produced by the magnetic bacterium.

  The present invention has been made in order to solve such a problem, wherein various biotinylated polypeptide sequences are fused with magnetic fine particle membrane proteins and expressed in magnetic bacteria, and magnetic fine particles are recovered. The presence or absence of a biotinylated label was confirmed. As a result, it was found that biotinylation was efficiently carried out in the microbial cells only when having a specific amino acid sequence. Furthermore, the inventors have found that peptide sequences that are not biotinylated in the microbial cells can be labeled with biotin in vitro after recovering the magnetic fine particles, and the present invention has been completed based on these findings.

  That is, in the first aspect, the method for producing a biotin-labeled magnetic fine particle of the present invention comprises a step of expressing a fusion protein of a magnetic particle membrane protein derived from a magnetic bacterium or a fragment thereof and a biotin-labeled sequence in the magnetic bacterium, Isolating magnetic microparticles from the magnetic bacteria. The biotin-labeled sequence consists of the amino acid sequence shown in SEQ ID NO: 2 (biotin carboxyl carrier protein: BCCP) or an amino acid sequence in which one or more amino acids are deleted, substituted, added and / or inserted in the amino acid sequence, And it is preferable that it can be biotinylated in magnetic bacteria. In one embodiment, the biotin-labeled sequence consists of amino acid residues at positions 72 to 149 of the amino acid sequence shown in SEQ ID NO: 2.

  In another embodiment, the biotin-labeled sequence is composed of a biotin-accepting peptide sequence (BAP), and the method further comprises contacting the isolated magnetic microparticles with E. coli or yeast biotin ligase. The biotin accepting peptide sequence is preferably any one selected from the amino acid sequences shown in SEQ ID NOs: 3 to 5.

  In still another embodiment, the fusion protein preferably contains both BCCP and BAP, and each is expressed at a predetermined position of the fusion protein on the surface of the magnetic fine particle film.

  In another aspect of the present invention, a fusion protein of a magnetic particle membrane protein Mms13 derived from magnetic bacteria or a fragment thereof and a biotin-labeled sequence containing at least amino acid residues 72 to 149 of the amino acid sequence shown in SEQ ID NO: 2 There are provided magnetic bacteria transformed with an expression vector encoding, and magnetic microparticles in which the fusion protein is expressed on a magnetic microparticle film. The fragment of the magnetic particle film protein Mms13 preferably contains at least 1 to 87 amino acid residues from the N-terminal in the amino acid sequence shown in SEQ ID NO: 6. In one embodiment, the fusion protein further comprises one or more biotin acceptor peptide sequences selected from the amino acid sequences shown in SEQ ID NOs: 3 to 5, wherein each of the biotin label sequence and the biotin acceptor peptide sequence is It is expressed at a predetermined position of the fusion protein on the surface of the magnetic fine particle film.

  According to the method of the present invention, a biotin-labeled sequence that can be biotinylated at a desired time after extraction from the microbial cells and from the microbial cells can be introduced into the magnetic microparticles using the production ability of magnetic bacteria. The method of labeling biotin inside the cell is more efficient than the method outside the cell, and biotin-labeled magnetic microparticles can be produced by a simple method that can be recovered from magnetic bacteria by magnetic separation.

[Definition]
As used herein, the term “magnetic bacterium” is a bacterium having the ability to accumulate magnetic microparticles in the body. Examples include, but are not limited to, Magnetospirillum species such as Magnetospirillum magneticum AMB-1, MS-1, and SR-1, and Desulfovibrio species such as Desulfovibrio sp. RS-1. These magnetic bacteria generate and hold chain-like magnetic particles called a magnetosome having a particle size of about 50 to 100 nm in the cells. These magnetic particles have characteristics such as high dispersibility, existence of a stable organic thin film, and single domain fine particles.

  In the present specification, the term “magnetic fine particle film protein” refers to a protein present in an organic film covering the magnetic fine particles of the magnetic bacteria. Various proteins existing on the cell membrane or on the magnetic fine particle membrane have been identified so far in magnetic bacteria. For example, there are Mms5, Mms6, Mms7, Mms13, and the like as magnetic fine particle membrane proteins derived from magnetic spiroram species magnetic bacteria, and the DNA base sequences encoding them are databases such as DDBJ and GenBank as access numbers AB096081 and AB096082. It is registered in.

  The terms “biotin-labeled sequence” or “biotinylated sequence” are those present in biotin enzymes such as acetyl-CoA carboxylase and transcarboxylase (enzymes that use biotin bound to a specific lysine residue as a coenzyme). An amino acid sequence that can be biotinylated by the action of biotin ligase. It is known that a biotin enzyme is biotinylated not only by biotin ligase derived from the same organism but also by biotin ligase derived from other species. This is considered to be because the three-dimensional structure of the biotinylation site is conserved across species. This conformational homology can be inferred from a comparison of the amino acid sequences of biotin enzymes derived from different organisms, but the sequence around the lysine residue undergoing biotinylation is particularly highly homologous, and many biotins A major feature of the enzyme is that sequence conservation on the C-terminal side is higher than that on the N-terminal side. The detailed mechanism by which biotin ligase recognizes these sequences has not yet been elucidated. Many biotin enzymes have a domain or subunit consisting of a biotin-labeled sequence, and these are referred to as “biotin carboxyl carrier protein (BCCP)”.

  The term “biotin-accepting peptide” is a kind of the above-mentioned biotin-labeled sequence, but BCCP is a relatively large polypeptide of biotin enzyme, and refers to a relatively small peptide of about 15 to 27 residues. This peptide was developed for use as a site-specific biotinylation tag that does not inhibit the function of the protein (see Non-Patent Document 2).

[Method for producing biotin-labeled magnetic fine particles]
The method for producing biotin-labeled magnetic fine particles of the present invention comprises (a) a step of expressing a fusion protein of a magnetic particle membrane protein derived from magnetic bacteria or a fragment thereof and a biotin-labeled sequence in magnetic bacteria, and (b) the magnetic bacteria. Isolating the magnetic microparticles from the process.

  In the step (a), an expression vector using a DNA encoding a magnetic particle membrane protein or a fragment thereof and a DNA encoding a biotin-labeled sequence is constructed, and this is introduced into a magnetic bacterium and fused in the magnetic bacterium. Express the protein. In this case, 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 promoter sequence.

  The promoter sequence is not particularly limited as long as it can be transcribed in the magnetic bacterium, but a promoter having high transcriptional activity can be obtained by identifying a protein present in a large amount in the magnetic bacterium and promoting the expression thereof. Can be obtained. For example, the promoter region of DNA encoding proteins Msp1 (mm10), Msp2 (mm24), Msp3 (mm1) and Mms16 (mms16) expressed on the magnetic microparticle membrane of the magnetic bacterium Magnetospirillum magneticumum AMB-1 Can be mentioned. These promoter sequences have already been reported by the present inventors (see, for example, JP-A-2006-75103), and the contents disclosed therein are incorporated herein by reference.

  In the present invention, the magnetic particle film protein or a fragment thereof is used as an anchor protein for presenting the protein on the magnetic fine particle film. As the anchor protein, a magnetic fine particle membrane protein (for example, Mms5, Mms6, Mms7, Mms13, etc.) derived from the magnetic bacterium Magnetospirillum magneticumum AMB-1 strain is preferably used. In particular, the Mms13 protein is preferable because it is firmly bound to the magnetic fine particles. Mms13 is a membrane twice-penetrating protein consisting of 124 amino acid residues, and it is considered that the N-terminal and C-terminal are localized on the surface of the magnetic fine particle film. Therefore, the fusion protein produced using the entire region of Mms13 can be anchored onto the cell membrane or the magnetic fine particle membrane. Mms13 has a region that is not a transmembrane site in the C-terminal region (amino acid residues at positions 88 to 124 of the amino acid sequence represented by SEQ ID NO: 6), and this region may not exist. . That is, it is sufficient if the amino acid sequence shown in SEQ ID NO: 6 contains at least 1 to 87 amino acid residues from the N-terminal.

  The biotin-labeled sequence in the method of the present invention is not particularly limited as long as it is a sequence that can be biotinylated by biotin ligase. Proteins that can be biotinylated in nature are rare, and only the acetyl-CoA carboxylase BCCP is known in E. coli. The number of proteins that can be biotinylated in eukaryotic cells is slightly higher, and yeast has 4 or 5 biotin proteins depending on the growth conditions. Therefore, it may be biotinylated by any biotin ligase existing in nature, but preferably a sequence that can be biotinylated in a magnetic bacterium. The biotin ligase of magnetic bacteria has not yet been isolated, but the presence of homologues of known biotin ligase is suggested from the genomic DNA sequence.

  In a preferred embodiment of the present invention, the biotin-labeled sequence consists of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are deleted, substituted, added and / or inserted in the amino acid sequence, It is a sequence that can be biotinylated by biotin ligase in magnetic bacteria. “One or more amino acids” refers to at most about 10 to 20% of the total number of amino acid residues, for example, about 1 to 30, preferably about 1 to 15, and more preferably about 1 to 10 Most preferably, the number is about 1 to 5. The biotin ligase activity can be measured by various methods well known to those skilled in the art. For example, incorporation of tritium-labeled biotin into a protein can be performed by measuring radioactivity.

  In the method of the present invention, the fusion protein is efficiently biotinylated by biotin ligase retained by magnetic bacteria. Magnetic particle membrane proteins of magnetic bacteria are synthesized in bacteria and then gather in the inner cell membrane. The inner membrane is depressed to form vesicles composed of lipid membranes, and then iron ions are formed in the vesicles. It is thought that magnetite is formed by accumulation. The biotin-labeled sequence fused with the magnetic particle membrane protein can be contacted with endogenous biotin ligase, for example, in the cytoplasm or at the stage of accumulation in the inner membrane, so that it is biotinylated after being localized on the magnetic particle membrane. Will be biotinylated with higher efficiency.

  In another embodiment of the present invention, the biotin-labeled sequence is at least 50%, preferably 70%, 80%, 85%, 90%, 97, with the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence. %, A sequence that can be biotinylated by biotin ligase of magnetic bacteria. The degree of protein homology can be expressed as a percentage of identity when the amino acid sequences of two proteins are properly aligned, and the rate of occurrence of exact matches between the sequences can be expressed as means. Appropriate alignment between sequences for identity comparison can be determined using various algorithms, such as the BLAST algorithm (Altschul SF J Mol Biol 1990 Oct 5; 215 (3): 403-10).

  Various methods known to those skilled in the art can be used as a method for preparing a variant of such a biotin-labeled sequence. For example, the base sequence encoding the target amino acid position is encoded by the amino acid to be modified. Using the primer substituted for the base sequence, the DNA substituted with the base sequence encoding the amino acid to be modified is amplified by PCR to obtain DNA encoding the biotin-labeled sequence variant, which is introduced into a magnetic bacterium. Can be expressed. Alternatively, it can be performed by a known site-directed mutagenesis method such as Kunkel method or Gapped duplex method, and a mutation introduction kit (for example, Mutan-K or Mutan-G) using these methods. (TAKARA etc.) can be used.

  In one embodiment of the present invention, the biotin-labeled sequence preferably consists of amino acid residues at positions 72 to 149 of the amino acid sequence shown in SEQ ID NO: 2. It is known that lysine residues of biotin-modified proteins are present in the amino acid sequence near the carboxyl terminus. From the homology of the peripheral sequences of lysine residues subjected to biotinylation, the biotinylation site in this embodiment is considered to be the 35th lysine residue from the carboxyl terminus.

  In other embodiments, the biotin labeling sequence described above may be a biotin accepting peptide sequence. Various peptide sequences that can be biotinylated by biotin ligase from E. coli or yeast have been reported. For example, peptides consisting of the amino acid sequences shown in SEQ ID NOs: 3 and 4 were screened as peptides that can be biotinylated by E. coli biotin ligase from a peptide library synthesized by linking to a lac repressor (non-non-reactive). Patent Document 2). In addition, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 was biotinylated by yeast biotin ligase using a phage display peptide library, but was found as a peptide sequence that was not biotinylated by E. coli biotin ligase. (Chen, I. et al., Journal of the American Chemical Society, (2007) Vol. 129, pp. 6619-6625). In this embodiment, these peptides can be expressed by being fused to the N-terminus or C-terminus of the anchor protein described above. Then, after isolating magnetic microparticles from magnetic bacteria, the microparticles may be contacted with E. coli or yeast biotin ligase in vitro. The conditions for in vitro biotinylation reactions are known, for example, E. coli biotin ligase requires ATP as an energy source.

  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. Therefore, in the step (b) in the method of the present invention, biotin-labeled magnetic fine particles can be easily obtained by destroying or dissolving the bacterial cells of magnetic bacteria and then collecting the magnetic fine particles using magnetism. A method of simply using magnetic fine particles from magnetic bacteria is well known in the art, and one specific example is shown in the examples described below.

[Biotin-labeled magnetic fine particles]
The biotin-labeled magnetic fine particles of the present invention are magnetic fine particles that can be obtained by the production method described above. That is, a preferred magnetic fine particle of the present invention is a magnetic particle film protein Mms13 derived from magnetic bacteria or a fragment thereof and a biotin-labeled sequence containing an amino acid residue at least positions 72 to 149 of the amino acid sequence shown in SEQ ID NO: 2. Magnetic fine particles obtained by expressing a fusion protein on a magnetic fine particle film. The Mms13 protein and the biotin-labeled sequence are as described above.

  In a preferred embodiment of the present invention, the fusion protein expressed on the magnetic fine particle film is a biotin-labeled sequence (BCCP) derived from the magnetic bacterium shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NOs: 3 to 5. One or more biotin acceptor peptide sequences (BAP) selected from the above, and each of the biotin label sequence and biotin acceptor peptide sequence is expressed at a predetermined position of the fusion protein on the surface of the magnetic fine particle film. is there. A method for constructing a fusion protein, specifically, a biotin-labeled sequence and a biotin-accepting peptide sequence can be linked to desired positions exposed on the surface of the magnetic fine particle in the Mms13 protein, whereby the distance between both biotin labels Can be controlled. For example, if biotin labeling can be performed at a position close to the surface of the magnetic fine particle film with time, it can be labeled with two different molecules that can interact on the magnetic fine particle film, and is produced by the method of the present invention. Various chemical or biological functions can be added to the magnetic fine particles.

  The present invention will be described in more detail by the following examples, but the scope of the present invention is not limited to these examples.

1. Experimental method 1-1. Reagents and instruments Biotin ligase derived from E. coli was purchased from AVIDITY, LLC. (Colorado, USA). Gold nanoparticle labeled streptavidin was purchased from Nanocs inc. TRITC labeled streptavidin was purchased from BECKMA COULTER. Alkaline phosphatase labeled streptavidin was purchased from CHEMICON International Inc. All other reagents were commercially available special grades for research or similar ones, and the reagents were prepared using distilled water and ultrapure water obtained by treating distilled water with MilliQ Lab of Japan Millipore Corporation. Luminometer Lucy-2 from Aloka Co., Ltd. was used for measuring the luminescence intensity. For fluorescence intensity measurement, a fluorescence spectrophotometer FluroMax-4 manufactured by HORIBA ITEC was used.

1-2. Construction of plasmid Biotin acceptor peptide (Biotin Acceptor Petide: BAP, amino acid sequence: MAQRLFHILDAQKIEWHGPKGGS: SEQ ID NO: 3), Biotin-Carboxyl Carrier Protein derived from magnetic bacteria (mBCCP; 149 amino acid residues, SEQ ID NO: 2), and C-terminal side of mBCCP A protein expression vector was constructed in which the 78 amino acid residue portion (mBCCP78) was fused with the magnetic bacterial particle membrane localized protein Mms13 (FIGS. 1 and 2).

  pUMGP16M13 is a plasmid in which a PCR product containing Pmms16 and mms13 gene downstream thereof is ligated to pUMG digested with SspI (see Applied and Environmentl Microbiology (2006) vol. 72 p465, method of construction of expression vectors).

  pUM13GFPBAP is a plasmid obtained by introducing a GFP gene into pUMGP16M13 digested with SspI and a BAP gene downstream thereof. Primer Fw: ATGGTGAGCAAGGGCGC (SEQ ID NO: 7) and a primer containing a BAP gene Rv: TTAAGAACCACCTTTCGGACCGTGCCATTCAATTTTCTGAGCGTCTTGAATGTGGAGCCATGCTGTG ) Was designed and PCR was performed using pAcGFP1 (Takara Bio) as a template. Ligation between the amplified fragment and pUMGP16M13 digested with SspI was performed, and pUM13GFPBAP was obtained using the obtained ligation sample (FIG. 1).

  pUM13GFP is a plasmid in which the GFP gene is introduced into pUMGP16M13 digested with SspI. In order to amplify the gene encoding GFP, primer Fw: 5′-GTGAGCAAGGGCGCCGAG-3 ′ (SEQ ID NO: 9) and primer Rv: 5′-TTACTTGTACAGCTCATC (SEQ ID NO: 10) were designed, and pAcGFP1 (Takara Bio) was used as a template. PCR was performed. Ligation was performed between the amplified fragment and pUMGP16M13 digested with SspI, and pUM13GFP was obtained using the obtained ligation sample.

  Primer NsiI-BAP-F: 5′-ATGCATATGGCTCAGCGTCTGTTCCA added with NsiI site (underlined sequence) for constructing pUMPAPM13GFP expressing protein fused with BAP at the N-terminus of Mms13 and green fluorescent protein GFP at the C-terminus -3 ′ (SEQ ID NO: 11) and NsiI-BAP-R: 5′-ATGCATAGAACCACCTTTCGGACCGT-3 ′ (SEQ ID NO: 12) were designed, and PCR was performed using pUM13GFPBAP as a template. The PCR product was subcloned into pCR4-Blunt-TOPO, and the sequence of the insert sequence was analyzed. Next, the insert sequence was excised using NsiI, and ligated with NsiI-digested plasmid pUM13GFP. The obtained ligation sample was used to transform E. coli DH5α. A plasmid was extracted from the obtained colonies, and the insertion direction of the insert sequence was confirmed by sequence analysis (FIG. 2).

  In order to construct pUM13BAP, pUM13mBCCP, and pUM13mBCCP78 that express proteins fused with BAP, mBCCP, and mBCCP78 at the C-terminus of Mms13, respectively, BAP-F: 5′-ATGGCTCAGCGTCTGTTCCA-3 ′ (SEQ ID NO: 13) , BAP-R: 5'-AGAACCACCTTTCGGACCGT-3 '(SEQ ID NO: 14), mBCCP-F: 5'-ATGGGCAACAAGACTCCCATC-3' (SEQ ID NO: 15), mBCCP78-F: 5'-CATCCCGGCGCGGTG as mBCCP and mBCCP78 amplification primers -3 ′ (SEQ ID NO: 16), mBCCP-R: 5′-CTATTCGATGATCAGCAAGGGC-3 ′ (SEQ ID NO: 17) was designed. PCR amplification of BAP was performed using pUM13GFPBAP as a template. Further, PCR amplification of mBCCP and mBCCP78 was performed using the magnetic bacterium Magnetospirillum magneticum AMB-1 genome as a template. Each PCR product was ligated with the plasmid pUMGP16M13 digested with SspI, and Escherichia coli DH5α was transformed with the obtained ligation sample. A plasmid was extracted from the obtained colonies, and the insertion direction of the insert sequence was confirmed by sequence analysis (FIG. 2).

1-3. Magnetic Bacterial Transformation, Culture and Preparation of BacMPs The wild-type magnetic bacterium Magnetospillum magneticum AMB-1 was inoculated into 4.5 liters of MSGM (Blakemore et al. 1979), and microaerobic by bubbling argon gas for 15 minutes. Then, the cells were statically cultured at room temperature for about 5 days. Moreover, the transformant introduced with plasmid pUMPAPM13GFP by electroporation was cultured in 5 μg / ml, ampicillin-containing MSGM, and the transformant introduced with pUM13mBCCP and pUM13mBCCP78 was cultured in MSGM containing 5 μg / ml ampicillin and 50 μM biotin.

The cultured cells are collected by centrifugation at 7200 rpm and 4 ° C. for 10 minutes, suspended in 30 ml of phosphate buffered saline (PBS, pH 7.4), and then 2000 kg / cm using a French press. ² and crushed. Thereafter, a neodymium-boron (Nd-B) magnet is attached to the bottom of the Erlenmeyer flask containing the bacterial cell disruption liquid to magnetically separate magnetic fine particles (BacMPs), and 2- [4-Hydroxyethyl] -1-piperazinyl] ethanesulfonic acid. Washed 10 times in (HEPES) buffer (10 mM, pH 7.4) using an ultrasonic washer. Washed BMPs were suspended in PBS and stored at 4 ° C.

1-4. Biotin labeling of BAP with E. coli-derived biotin ligase 50 μg of BacMPs was suspended in 50 mM MgCl ₂ , 10 μM biotin, 100 mM ATP, and 10 μg / ml E. coli-derived biotin ligase-containing PBS (pH 7.4, 100 μl). Stir for hours. Thereafter, the magnetically separated BacMPs were washed 3 times with 50 μl of PBS.

1-5. Immobilization of streptavidin to biotin-labeled BacMPs 100 μl of 20 μg / ml TRITC-labeled streptavidin (TRITC-SA) was added to 10 μg of BacMPs extracted from the magnetic bacterium M. magneticum AMB-1 transformant at room temperature for 1 hour. Stir. Thereafter, the plate was washed 3 times with 100 μl PBST, and BacMPs were suspended in 400 μl PBST and observed with a fluorescence microscope.

For BacMPs (50 μg) extracted from the magnetic bacterium M. magneticum AMB-1 transformant, 1/100 diluted alkaline phosphatase labeled streptavidin (ALP-SA) solution (protein concentration 5.4 mg / ml, 2 mg / ml BSA) 100 μl) was added and stirred at room temperature for 1 hour. After washing 3 times with 100 μl PBST, BacMPs were suspended in 50 μl PBS, lumiphos 530 (3.3 × 10 ⁻⁴ mol / l, 50 μl) was added, and the luminescence intensity after 5 minutes was measured.

  In order to measure the amount of streptavidin that can be immobilized on BacMPs, TRITC-labeled streptavidin was immobilized, and the fluorescence intensity was measured. 200 μl of TRITC labeled streptavidin solution was added to 100 μg of BacMPs and stirred at room temperature for 1 hour. Thereafter, the plate was washed 3 times with 200 μl of PBST, and 50 μg of BacMPs was suspended in 500 μl of PBS. Thereafter, the fluorescence intensity of the particles (excitation wavelength: 545 nm, fluorescence wavelength: 580 nm) was measured using a fluorescence spectrophotometer. To prepare a calibration curve, a TRITC solution (500 μl) with a known concentration was added to 50 μg of wild strain-derived BaMPs (WT-BacMPs), and the fluorescence intensity was measured in the same manner.

1-6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis 2 mg of magnetic bacterial particles (BacMPs) extracted from the magnetic bacterium Magnetospirillum magneticum AMB-1 are suspended in 1% sodium dodecyl sulfate (SDS) solution and boiled at 100 ° C. for 30 minutes. BacMPs membrane protein was extracted. The extracted membrane protein was mixed with SDS sample buffer (final concentration: 6.25 mM Tris-HCl [pH6.8], 5% 2-mercaptoethanol, 2% SDS, 5% sucrose and 0.002% bromophenol blue), and 12.5% (wt / vol) Electrophoresis was performed using polyacrylamide gel. Thereafter, the gel was stained with cumin brilliant blue R-250.

1-7. Immobilization of Streptavidin to Biotin-Labeled BacMPs In order to confirm that streptavidin could be immobilized on BacMPs, gold nanoparticle-labeled streptavidin was immobilized and observed with a transmission electron microscope. 200 μl of gold nanoparticle labeled streptavidin solution diluted 1/2 with PBST was added to 10 μg of BacMPs and stirred at room temperature for 1 hour. Then, it was washed 3 times with 100 μl PBST and once with 100 μl ultrapure water. After washing, BacMPs were suspended in ultrapure water and dried on a grid for 30 minutes. Thereafter, the particles were observed using a transmission electron microscope (H700H, Hitachi, Ltd.).

2. Result 2-1. In vivo biotin labeling of mBCCP (78) -presented BacMPs Magnetic microparticles (mBCCP-BacMPs) were extracted from pUM13mBCCP transformants, and the presence or absence of biotin labeling was confirmed using TRITC-SA. As a result, it was observed that TRITC-SA was immobilized on mBCCP-BacMPs. From this, it was shown that mBCCP presented on BacMPs is labeled with biotin in the magnetic bacteria (FIG. 3).

  Next, we attempted to shorten the biotin-labeled tag, mBCCPA, displayed on BacMPs. In general, it is known that among amino acid sequences possessed by BCCP, sequences involved in biotinylation are concentrated on the C-terminal side. Therefore, the amino acid sequence of mBCCP derived from magnetic bacteria M. magneticum AMB-1 was compared with BCCP derived from closely related species, M. magnetotacticum MS-1, M. gryphiswaldense MSR-1, and Rhodospirirum rubrum (FIG. 4). As a result, it was found that the common sequence was concentrated in the portion corresponding to the C-terminal side 78 residues (mBCCP78) of the 72nd to 149th residues. Therefore, M. magneticum AMB-1 was transformed with plasmid pUM13mBCCP78 expressing a protein in which mBCCP78 was fused to the C-terminus of Mms13, and magnetic particles (mBCCP78-BacMPs) were extracted.

  When SA-ALP was used to confirm whether this mBCCP78-BacMPs was labeled with biotin in the magnetic bacteria body, it showed a luminescence intensity comparable to that of mBCCP-BacMPs (FIG. 5). From this, it was shown that mBCCP78 having only 78 amino acid residues on the C-terminal side among mBCCP composed of 149 amino acid residues is also recognized by biotin ligase and labeled with biotin in the magnetic bacteria.

2-2. In vitro biotin labeling of BAP-presented BacMPs In order to confirm that BAP expressed on BacMPs is biotinylated by the action of E. coli-derived biotin ligase, biotin ligase treatment or untreated BAP-Mm13-GFP-expressing BacMPs and SA-ALP was added to Mm13-GFP expressing BacMPs. As a result, high luminescence intensity was obtained only in BAP-Mm13-GFP-expressing BacMPs treated with biotin ligase (FIG. 6). From this, it was shown that BAP on BacMPs can be specifically labeled with biotin in vitro using biotin ligase. Moreover, since ALP-SA did not bind to biotin ligase-untreated BAP-Mm13-GFP-expressing BacMPs, it was shown that BAP was not biotinylated in AMB-1.

  In addition, the vertical axis | shaft of the graph shown in FIG.5 and FIG.6 represents the activity of streptavidin labeled alkaline phosphatase by the fluorescence intensity. Although the amount of magnetic fine particles (BacMPs) used is the same, the fluorescence intensity differs by about 10 times, so that the biotinylation reaction in magnetic bacteria is significantly more efficient than the in vitro biotinylation reaction. I understand that there is.

2-3. Confirmation of Mms13-mBCCP fusion protein expression on BacMPs
Extracting the membrane protein on BacMPs obtained from M. magneticum AMB-1 wild type strain and the plasmid pUM13mBCCP transformant expressing the fusion protein of membrane protein Mms13 on BacMPs and AMB-1-derived biotin-binding protein mBCCP; SDS-PAGE was performed. The result is shown in FIG. In BacMPs (mBCCP-BacMPs) obtained from the pUM13mBCCP transformant, a prominent band was obtained around 30 to 43 kDa. Since the Mms13-mBCCP fusion protein is a protein of about 31 kDa, the band obtained by SDS-PAGE was considered to be Mms13-mBCCP. From the above results, it was confirmed that the Mms13-mBCCP fusion protein was introduced at high expression on BacMPs.

2-4. Observation of gold nanoparticles immobilized on BacMPs In order to confirm that streptavidin was introduced on BacMPs, gold nanoparticle-labeled streptavidin was added to WT-BacMPs and mBCCP-BacMPs, and a transmission electron microscope was added. Observed at. The result is shown in FIG. It was confirmed that more gold nanoparticles were immobilized on mBCCP-BacMPs than WT-BacMPs. This showed that mBCCP presented on BacMPs was biotinylated and that streptavidin could be immobilized thereon.

2-5. Measurement of the amount of TRITC-labeled streptavidin immobilized on BacMPs In order to measure the amount of streptavidin that can be immobilized on BacMPs, TRITC-labeled streptavidin was added to WT-BacMPs and mBCCP-BacMPs, and the fluorescence intensity was measured. The result is shown in FIG. It was shown that about 1300 ng streptavidin can be immobilized on 1 mg mBCCP-BacMPs. This indicates that about 15 molecules of streptavidin (60 kDa) can be immobilized on one particle of BacMPs (particle size: 75 nm, density: approximated to 5.2 g / cm ³ ).

It is a schematic diagram which shows the construction method of the expression vector in magnetic bacteria. It is a schematic diagram which shows the construction method of the fusion protein expression plasmid used for the method of this invention. It is a microscope observation image after adding TRITC labeled streptavidin to the magnetic fine particles produced by the method of the present invention and washing. (Scale bar: 10 μm) This is an alignment comparing the amino acid sequences of BCCP and BAP derived from Magnetospirillum magneticum AMB-1 and its related species. It is the result of investigating whether or not the fusion protein expressed in magnetic bacteria is biotinylated in vivo. It is the result of confirming in vitro biotin labeling of magnetic microparticles using streptavidin labeled alkaline phosphatase. The result of SDS-PAGE of the membrane protein extracted from WT-BacMPs and mBCCP-BacMPs is shown. It is a transmission electron microscope observation image after adding gold nano labeling streptavidin to WT-BacMPs and mBCCP-BacMPs, and washing. FIG. 2 is a binding saturation curve of TRITS-labeled streptavidin to WT-BacMPs and mBCCP-BacMPs.

Claims (11)

A step of expressing a fusion protein of a magnetic particle membrane protein derived from magnetic bacteria or a fragment thereof and a biotin-labeled sequence in the magnetic bacteria,
Isolating magnetic microparticles from the magnetic bacteria;
A method for producing biotin-labeled magnetic fine particles, comprising:   The biotin-labeled sequence consists of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are deleted, substituted, added and / or inserted in the amino acid sequence, and is biotinylated in a magnetic bacterium. The method of claim 1 wherein:   The method according to claim 1 or 2, wherein the biotin-labeled sequence comprises amino acid residues at positions 72 to 149 of the amino acid sequence shown in SEQ ID NO: 2.   The method according to claim 1, further comprising the step of bringing the biotin-labeled sequence into a biotin-accepting peptide sequence and bringing the isolated magnetic microparticles into contact with E. coli or yeast biotin ligase.   The method according to claim 4, wherein the biotin-accepting peptide sequence is any one selected from the amino acid sequences shown in SEQ ID NOs: 3 to 5.   The fusion protein further includes one or a plurality of biotin acceptor peptide sequences selected from the amino acid sequences shown in SEQ ID NOs: 3 to 5, and each of the biotin label sequence and the biotin acceptor peptide sequence is the surface of the magnetic fine particle film. The method according to claim 2 or 3, wherein the method is expressed at a predetermined position of the fusion protein.   Transformation with an expression vector encoding a fusion protein of a magnetic particle membrane protein Mms13 derived from magnetic bacteria or a fragment thereof and a biotin-labeled sequence containing at least amino acid residues 72 to 149 of the amino acid sequence shown in SEQ ID NO: 2. A magnetic bacterium.   A fusion protein of magnetic particle membrane protein Mms13 derived from magnetic bacteria or a fragment thereof and a biotin-labeled sequence containing at least amino acids 72 to 149 of the amino acid sequence shown in SEQ ID NO: 2 is expressed on the magnetic particle membrane Magnetic fine particles.   The magnetic fine particle according to claim 8, wherein the fragment of the magnetic particle film protein Mms13 contains at least 1 to 87 amino acid residues from the N-terminal in the amino acid sequence shown in SEQ ID NO: 6.   The fusion protein further includes one or a plurality of biotin acceptor peptide sequences selected from the amino acid sequences shown in SEQ ID NOs: 3 to 5, and each of the biotin label sequence and the biotin acceptor peptide sequence is the surface of the magnetic fine particle film. The magnetic fine particle according to claim 8 or 9, which is expressed at a predetermined position of the fusion protein.   The magnetic fine particle according to claim 10, wherein the biotin-labeled sequence and the biotin-accepting peptide sequence are expressed by being fused to the C-terminus and N-terminus of the magnetic particle membrane protein Mms13 or a fragment thereof, respectively.