JP2009118802A - Method for producing biotin-labeled magnetic fine particles - Google Patents
Method for producing biotin-labeled magnetic fine particles Download PDFInfo
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
本発明は、ビオチン標識された磁気微粒子の製造方法に関し、さらに詳細には、磁性細菌内にて磁気粒子膜タンパク質と、ビオチン標識配列との融合タンパク質を発現させることにより、磁性細菌が生産する粒径約50〜150nmの磁気微粒子上に簡便にビオチン標識する方法及び当該方法により製造された磁気微粒子等に関する。 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.
近年、バイオセンサやイムノセンサ用のタンパク質固定化担体として磁気微粒子が利用されている。磁気微粒子は磁気による回収が容易であるため、イムノアッセイのみならず、ドラッグキャリア、細胞分離技術等の幅広い分野への応用も期待されている。イムノアッセイ等における物質の標識方法の1つとして、アビジン又はストレプトアビジンと、ビオチンとの間の結合がある。これらの結合は極めて安定である(解離定数10−15M)ことから、検出感度の向上や迅速なアッセイを行なうために有用である。例えば、検体中の抗原を測定するイムノアッセイの場合、抗原に結合したビオチン化抗体を、標識したアビジンまたはストレプトアビジンと反応させて、洗浄後、残存した標識強度を測定することが行われている。 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.
抗体等のビオチン化のために、被ビオチン化物質のアミノ基、カルボキシル基、チオール基(スルフヒドリル基またはメルカプト基)などを利用してビオチンを固定化できるビオチン化試薬が種々市販されている。しかし、これらの化学試薬はタンパク質の活性化部位にも無差別に結合してしまうため、タンパク質のもつ機能を阻害してしまうという問題点があった。そのため、タンパク質の機能を阻害しない、部位特異的な標識技術の開発が望まれていた。 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.
細胞内でビオチンリガーゼによって認識されるタンパク質ドメインは、生物種を越えて広く保存されており、このタンパク質ドメインと融合させた種々のタンパク質が生体内でビオチン化されることが報告されている(例えば、非特許文献1参照)。しかし、このビオチン化されうるドメインは、最小でも75アミノ酸残基を必要とする大きなポリペプチドである。小さな部位特異的ビオチン化タグを開発するために、種々のビオチン酵素のビオチン周辺配列を模倣して15〜27残基のランダムなアミノ酸配列をもつペプチドライブラリーを構築し、大腸菌内でビオチン化するペプチド配列が探索された。ビオチン受容ペプチドと名づけられた比較的短いペプチドは、大腸菌のビオチンリガーゼにより効率的にビオチン化されることが見出された(例えば、非特許文献2参照)。 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).
一方、磁性細菌の菌体内において産生される磁気微粒子が知られている。この磁気微粒子又はその菌体自体は、磁石を用いることにより溶液から容易に分離することが可能であるため、タンパク質の製造及び単離、並びに種々の物質の回収、探索、検出及び定量に有用である。この磁気微粒子は、磁気微粒子膜と称されるリン脂質を主成分とする有機膜で被膜されており、この膜には種々の膜タンパク質が存在する(例えば、特許文献1〜3参照)。また、この膜タンパク質をアンカータンパク質として目的ポリペプチドとの融合タンパク質を磁気微粒子の表面で発現させる方法も報告されている(例えば、特許文献4参照)。 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).
ナノメートルスケールの磁性微粒子を効率的にビオチン標識するためには、磁気微粒子表面を被覆するポリマーにアミノ基等の官能基を導入しなければならず、作成工程が煩雑な上高価な試薬を用いることから、市販されているビオチン標識磁気微粒子は極めて高価である。また、磁性細菌の産生する磁気微粒子膜上に、上述のビオチン受容ペプチドを発現させた場合に磁性細菌内でビオチン化が起こるかどうかは未だ明らかではない。 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.
本発明は、かかる課題を解決するためになされたものであって、磁性微粒子膜タンパク質に種々のビオチン化されうるポリペプチド配列を融合させて磁性細菌内で発現させ、そして磁気微粒子を回収してビオチン化標識の有無を確認した。その結果、特定のアミノ酸配列を有する場合にのみ菌体内で効率的にビオチン化されることを発見した。さらに菌体内でビオチン化されないペプチド配列が、磁気微粒子を回収した後、インビトロにおいてビオチン標識しうることを見出し、これらの知見に基づいて本発明を完成した。 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.
すなわち、第一の視点において本発明のビオチン標識磁気微粒子の製造方法は、磁性細菌由来の磁気粒子膜タンパク質又はその断片と、ビオチン標識配列との融合タンパク質を、磁性細菌内で発現させる工程と、前記磁性細菌から磁気微粒子を単離する工程と、を含むことを特徴とする。前記ビオチン標識配列は、配列番号2に示したアミノ酸配列(ビオチンカルボキシル運搬タンパク質:BCCP)又は当該アミノ酸配列において1又は複数のアミノ酸が欠失、置換、付加及び/又は挿入されたアミノ酸配列からなり、かつ磁性細菌内でビオチン化されうることが好ましい。1つの実施形態において、前記ビオチン標識配列は、配列番号2に示したアミノ酸配列の72位〜149位のアミノ酸残基からなることを特徴とする。 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.
他の実施形態において、前記ビオチン標識配列がビオチン受容ペプチド配列(BAP)からなり、かつ単離された磁気微粒子と大腸菌又は酵母のビオチンリガーゼとを接触させる工程をさらに含むことを特徴とする。前記ビオチン受容ペプチド配列は、配列番号3〜5に示したアミノ酸配列から選択される何れかであることが好ましい。 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.
さらに異なる実施形態において、前記融合タンパク質は、前記BCCPとBAPの両方を含み、夫々が磁気微粒子膜表面において前記融合タンパク質の所定の位置に発現することが好ましい。 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.
本発明の他の視点において、磁性細菌由来の磁気粒子膜タンパク質Mms13又はその断片と、配列番号2に示したアミノ酸配列の少なくとも72位〜149位のアミノ酸残基を含むビオチン標識配列との融合タンパク質をコードする発現ベクターで形質転換されてなる磁性細菌、並びに当該融合タンパク質が磁気微粒子膜上に発現されてなる磁気微粒子が提供される。前記磁気粒子膜タンパク質Mms13の断片は、配列番号6に示したアミノ酸配列のうち、N末端から少なくとも1〜87個のアミノ酸残基を含むことが好ましい。1つの実施形態において、前記融合タンパク質が、配列番号3〜5に示したアミノ酸配列から選択される1又は複数のビオチン受容ペプチド配列をさらに含み、前記ビオチン標識配列及びビオチン受容ペプチド配列の夫々は、磁気微粒子膜表面において前記融合タンパク質の所定の位置に発現することを特徴とする。 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.
本発明の方法によれば、磁性細菌の生産能を利用して、菌体内及び菌体から抽出後の所望の時期にビオチン化されうるビオチン標識配列を磁気微粒子に導入することができる。菌体内でビオチン標識する方法は菌体外における方法に比べて効率的であり、磁性細菌から磁気分離により回収するだけの簡便な方法でビオチン標識磁気微粒子を製造することができる。 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.
[定義]
本明細書において、用語「磁性細菌」とは、体内に磁気微粒子を蓄積する能力を有する細菌である。例えば、Magnetospirillum magneticum AMB-1, MS-1, 及びSR-1等のマグネトスピリラム種の微生物、並びにDesulfovibrio sp. RS-1等のデサルフォビブリオ種の微生物が挙げられるがこれらに限定されない。これらの磁性細菌は、マグネトソームとよばれる50〜100nm程度の粒径をもつチェーン状の磁性粒子を菌体内に生成,保持している。これらの磁性粒子は高い分散性,安定な有機薄膜の存在,単磁区微粒子といった特性を有する。
[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.
本明細書において、用語「磁気微粒子膜タンパク質」とは、上記磁性細菌の磁気微粒子を被覆する有機膜に存在するタンパク質をいう。磁性細菌において、これまでに細胞膜上又は磁気微粒子膜上に存在する種々のタンパク質が同定されている。例えば、マグネトスピリラム種の磁性細菌に由来する磁気微粒子膜タンパク質として、Mms5、Mms6、Mms7及びMms13等があり、これらをコードするDNAの塩基配列はアクセス番号AB096081及びAB096082としてDDBJやGenBank等のデータベースに登録されている。 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.
用語「ビオチン標識配列」ないし「ビオチン化配列」とは、アセチル−CoAカルボキシラーゼやトランスカルボキシラーゼのようなビオチン酵素(特定のリジン残基に結合したビオチンを補酵素として利用する酵素)に存在するような、ビオチンリガーゼの働きによってビオチン化されうるアミノ酸配列をいう。ビオチン酵素は同じ生物由来のビオチンリガーゼだけでなく、他種生物由来のビオチンリガーゼによってもビオチン化されることが知られている。これはビオチン化部位の立体構造が種を越えて保存されているためであると考えられる。この立体構造の相同性は、異なる生物に由来するビオチン酵素のアミノ酸配列の比較から推測することができるが、ビオチン化を受けるリジン残基周辺の配列が特に相同性が高いこと、及び多くのビオチン酵素においてN末端側に比べてC末端側の配列保存性が高いことが大きな特徴である。ビオチンリガーゼがこれらの配列を認識する詳細な機構は未だ解明されていない。なお、多くのビオチン酵素は、ビオチン標識配列からなるドメイン又はサブユニットを有し、これらを「ビオチンカルボキシル運搬タンパク質:BCCP(Biotin Carboxyl Carrier Protein)」と称する。 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)”.
用語「ビオチン受容ペプチド」とは、上記ビオチン標識配列の一種であるが、BCCPがビオチン酵素の比較的大きなポリペプチドであるのに対し、15〜27残基程度の比較的小さなペプチドをいう。このペプチドは、タンパク質の機能を阻害しない部位特異的ビオチン化タグとして利用するために開発された(非特許文献2参照)。 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).
[ビオチン標識磁気微粒子の製造方法]
本発明のビオチン標識磁気微粒子の製造方法は、(a)磁性細菌由来の磁気粒子膜タンパク質又はその断片とビオチン標識配列との融合タンパク質を磁性細菌内で発現させる工程と、(b)前記磁性細菌から磁気微粒子を単離する工程と、を含む。
[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.
上記工程(a)は、磁気粒子膜タンパク質又はその断片をコードするDNAと、ビオチン標識配列をコードするDNAとを用いた発現ベクターを構築し、これを磁性細菌に導入して磁性細菌内で融合タンパク質を発現させる。この場合、上記発現ベクターは、磁性細菌内で転写制御可能なDNA断片をプロモーターとして含み、かつこのプロモーター配列の下流に融合タンパク質をコードするDNAを挿入する。 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.
このプロモーター配列は、磁性細菌内で転写可能であれば特に限定されないが、磁性細菌内で多量に存在するタンパク質を同定し、その発現を推進するプロモーター配列を使用することにより、転写活性の高いプロモーターを取得することができる。例えば、磁性細菌マグネトスピリラム・マグネティカムAMB−1株の磁気微粒子膜上で発現されているタンパク質Msp1(mm10)、Msp2(mm24)、Msp3(mm1)及びMms16(mms16)をコードするDNAのプロモーター領域の配列を挙げることができる。これらのプロモーター配列は、本発明者らによりすでに報告されており(例えば、特開2006−75103号公報参照)、これらに開示された内容は引用により本願明細書に組み込まれる。 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.
本発明において、磁気粒子膜タンパク質又はその断片は、磁気微粒子膜上にタンパク質を提示するためのアンカータンパク質として使用される。アンカータンパク質としては、磁性細菌マグネトスピリラム・マグネティカムAMB−1株に由来する磁気微粒子膜タンパク質(例えば、Mms5、Mms6、Mms7、及びMms13等)が好適に用いられる。特に、Mms13タンパク質は、磁気微粒子に強固に結合しているため好適である。Mms13は、124アミノ酸残基からなる膜2回貫通型タンパク質であり、N末端及びC末端が磁性微粒子膜表面に局在していると考えられている。従って、Mms13の全領域を用いて生成した融合タンパク質は、細胞膜上又は磁性微粒子膜上へのアンカリングが可能である。また、Mms13は、そのC末端領域に膜貫通部位でない領域(配列番号6で示されるアミノ酸配のうち、88〜124位のアミノ酸残基)を有し、この領域は存在していなくてもよい。すなわち、配列番号6に示したアミノ酸配列のうち、N末端から少なくとも1〜87個のアミノ酸残基を含んでいればよい。 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.
本発明の方法におけるビオチン標識配列は、ビオチンリガーゼによってビオチン化されうる配列であれば特に限定されない。自然界においてビオチン化されうるタンパク質は稀であり、大腸菌ではアセチル−CoAカルボキシラーゼのBCCPのみが知られている。真核細胞においてビオチン化されうるタンパク質の数はもう少し多く、酵母では増殖条件に依存して4個又は5個のビオチンタンパク質を有する。従って、自然界に存在する何れかのビオチンリガーゼによってビオチン化されればよいが、好ましくは磁性細菌内においてビオチン化されうる配列である。磁性細菌のビオチンリガーゼは、未だ単離されていないが、ゲノムDNA配列から公知のビオチンリガーゼの相同体の存在が示唆されている。 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.
本発明の好ましい実施形態において、上記ビオチン標識配列は、配列番号2に示したアミノ酸配列又は当該アミノ酸配列において1又は複数のアミノ酸が欠失、置換、付加及び/又は挿入されたアミノ酸配列からなり、かつ磁性細菌内でビオチンリガーゼによりビオチン化されうる配列である。「1又は複数のアミノ酸」とは、全長アミノ酸残基数の多くとも10〜20%程度をいい、例えば、1〜30個程度、好ましくは1〜15個程度、より好ましくは1〜10個程度、最も好ましくは1〜5個程度である。ビオチンリガーゼ活性は、当業者に周知の種々の方法により測定することができ、例えば、トリチウム標識されたビオチンのタンパク質への取り込みを、放射活性の測定により行なうことができる。 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.
本発明の方法において、前記融合タンパク質は、磁性細菌が保持しているビオチンリガーゼによって効率的にビオチン化される。磁性細菌の磁気粒子膜タンパク質は、細菌内で合成された後、細胞内膜に集まり、この内膜が陥没して脂質膜で構成された小胞が形成され、その後小胞内に鉄イオンが集積されることでマグネタイトが形成されると考えられる。この磁気粒子膜タンパク質と融合した上記ビオチン標識配列は、例えば細胞質内で、又は内膜に集積した段階で内在性のビオチンリガーゼと接触できるため、磁気粒子膜上に局在した後にビオチン化されるよりも高い効率でビオチン化されるのであろう。 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.
本発明の他の実施形態において、上記ビオチン標識配列は、配列番号2に示したアミノ酸配列からなるタンパク質又は当該アミノ酸配列と少なくとも50%、好ましくは70%、80%、85%、90%、97%、98%以上の相同性を有するタンパク質からなり、かつ磁性細菌のビオチンリガーゼによりビオチン化されうる配列である。タンパク質の相同性(ホモロジー)の程度は、2つのタンパク質のアミノ酸配列同士を適切に整列(アライメント)したときの同一性のパーセント値で表わすことができ、当該配列間の正確な一致の出現率を意味する。同一性比較のための配列間での適切な整列は種々のアルゴリズム、例えば、BLASTアルゴリズムを用いて決定することができる(Altschul SF J Mol Biol 1990 Oct 5; 215(3):403-10)。 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).
このようなビオチン標識配列の変異体を作製する方法としては、当業者に公知の種々の方法を用いることができ、例えば、目的のアミノ酸の位置をコードする塩基配列を改変すべきアミノ酸をコードする塩基配列に置換したプライマーを用いて、改変すべきアミノ酸をコードする塩基配列に置換したDNAをPCRにより増幅させてビオチン標識配列変異体をコードするDNAを取得し、これを磁性細菌に導入して発現させることができる。あるいは、Kunkel法又はギャップ二重鎖(Gapped duplex)法等の公知の部位特異的突然変異導入方法によって行うことができ、これらの手法を利用した変異導入用キット(例えばMutan−KやMutan−G(TAKARA)等)を利用することができる。 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.
本発明の1つの実施形態において、前記ビオチン標識配列は、配列番号2に示したアミノ酸配列の72位〜149位のアミノ酸残基からなることが好ましい。ビオチン修飾されるタンパク質のリジン残基は、カルボキシル末端の近くのアミノ酸配列中に存在することが知られている。ビオチン化を受けるリジン残基の周辺配列の相同性から、本実施形態におけるビオチン化部位はカルボキシル末端から35番目のリジン残基であると考えられる。 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.
他の実施形態において、上述したビオチン標識配列はビオチン受容ペプチド配列であってもよい。大腸菌や酵母のビオチンリガーゼによってビオチン化されうる種々のペプチド配列が報告されている。例えば、配列番号3及び4に示したアミノ酸配列からなるペプチドは、lacリプレッサーに連結して合成されたペプチドライブラリーから大腸菌のビオチンリガーゼによりビオチン化されうるペプチドとしてスクリーニングされたものである(非特許文献2参照)。また、配列番号5に示したアミノ酸配列からなるペプチドは、ファージディスプレイペプチドライブラリーを用いて、酵母のビオチンリガーゼによってビオチン化されるが、大腸菌のビオチンリガーゼではビオチン化されないペプチド配列として見出された(Chen, I. et al., Journal of the American Chemical Society, (2007) Vol.129, pp.6619-6625)。本実施形態においては、これらのペプチドを上述したアンカータンパク質のN末端又はC末端に融合させて発現することができる。そして、磁性細菌から磁性微粒子を単離した後に、インビトロで当該微粒子と大腸菌又は酵母のビオチンリガーゼとを接触させればよい。インビトロにおけるビオチン化反応の条件は公知であり、例えば、大腸菌のビオチンリガーゼは、エネルギー源としてのATPを必要とする。 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.
本発明の発現ベクターに融合タンパク質をコードする遺伝子を組み込んで磁性細菌を形質転換し、得られた形質転換体を適切な条件下で培養すると、菌体内に磁気微粒子が産生され、磁気微粒子を被覆している磁性微粒子膜にアンカリングした形で融合タンパク質が発現する。従って、本発明の方法における工程(b)は、磁性細菌の菌体を破壊又は溶解した後、磁気を利用して磁気微粒子を回収することにより、ビオチン標識磁気微粒子を容易に得ることができる。磁性細菌から磁気微粒子を単利する方法は、当該技術分野においてよく知られており、また、後述の実施例において1つの具体例が示される。 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.
[ビオチン標識磁気微粒子]
本発明のビオチン標識磁気微粒子は、上述した製造方法により得ることができる磁気微粒子である。すなわち、本発明の好ましい磁気微粒子は、磁性細菌由来の磁気粒子膜タンパク質Mms13又はその断片と、配列番号2に示したアミノ酸配列の少なくとも72位〜149位のアミノ酸残基を含むビオチン標識配列との融合タンパク質が磁気微粒子膜上に発現されてなる磁気微粒子である。Mms13タンパク質及びビオチン標識配列については、上述したとおりである。
[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.
本発明の好ましい実施形態において、磁気微粒子膜上に発現される融合タンパク質は、上述した配列番号2に示した磁性細菌由来のビオチン標識配列(BCCP)と、配列番号3〜5に示したアミノ酸配列から選択される1又は複数のビオチン受容ペプチド配列(BAP)とを同時に含み、前記ビオチン標識配列及びビオチン受容ペプチド配列の夫々は、磁気微粒子膜表面において前記融合タンパク質の所定の位置に発現するものである。融合タンパク質の構築方法、具体的にはMms13タンパク質において磁気微粒子表面に露出する所望の位置に、ビオチン標識配列とビオチン受容ペプチド配列とを夫々連結することができ、これによって両者のビオチン標識間の距離を制御することが可能となる。例えば、磁気微粒子膜表面の近接した位置に時間をおいてビオチン標識することができれば、磁気微粒子膜上で相互作用しうる2種類の異なる分子で標識することができ、本発明の方法により製造された磁気微粒子に様々な化学的又は生物学的な機能を付加することができるようになる。 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.
本発明は、以下の実施例によりさらに詳細に説明されるが、本発明の範囲はこれらの実施例に限定されるものではない。 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.実験方法
1−1.試薬及び器具
大腸菌由来ビオチンリガーゼはAVIDITY,LLC.(Colorado,USA)から購入した。金ナノ粒子標識ストレプトアビジンはNanocs inc.から購入した。TRITC標識ストレプトアビジンはBECKMA COULTERから購入した。アルカリホスファターゼ標識ストレプトアビジンはCHEMICON International Inc.から購入した。その他の試薬類は全て研究用の市販特級品またはそれに準じたものを用い、試薬等の調製は蒸留水及び蒸留水を日本ミリポア株式会社のMilliQ Labで処理した超純水を用いた。発光強度測定にはアロカ株式会社のルミノメーターLucy-2を利用した。蛍光強度測定にはホリバ株式会社ホリバアイテックの蛍光分光光度計 FluroMax-4を利用した。
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.プラスミドの構築
ビオチン受容ペプチド(Biotin Acceptor Petide:BAP、アミノ酸配列:MAQRLFHILDAQKIEWHGPKGGS:配列番号3)、磁性細菌由来Biotin-Carboxyl Carrier Protein (mBCCP;149アミノ酸残基、配列番号2)、及びmBCCPのC末端側78アミノ酸残基部分(mBCCP78)をそれぞれ磁性細菌粒子膜局在タンパク質Mms13と融合したタンパク質発現ベクターを構築した(図1及び2)。
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は、SspI消化したpUMGにPmms16、及びその下流にmms13遺伝子を含むPCR産物をライゲーションしたプラスミドである(Applied and Environmentl Microbiology (2006) vol.72 p465, Methodのconstruction of expression vectors、参照)。 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は、SspI消化したpUMGP16M13にGFP遺伝子、及びその下流にBAP遺伝子を導入したプラスミドである。ビオチンアクセプターペプチド(Biotin Acceptor petide:BAP)を融合したGFPをコードする遺伝子(789bp)を増幅するため、プライマーFw:ATGGTGAGCAAGGGCGC(配列番号7)、及びBAP遺伝子を含むプラーマーRv:TTAAGAACCACCTTTCGGACCGTGCCATTCAATTTTCTGAGCGTCCAGAATGTGGAACAGACGCTGAGCCATCTTGTACAGCTCATCCATGC(配列番号8)を設計し、pAcGFP1(タカラバイオ)を鋳型として用いてPCRを行った。増幅断片とSspI消化したpUMGP16M13とのライゲーションを行い、得られたライゲーションサンプルを用いてpUM13GFPBAPを取得した(図1)。 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は、SspI消化したpUMGP16M13にGFP遺伝子を導入したプラスミドである。GFPをコードする遺伝を増幅するため、プライマーFw:5’-GTGAGCAAGGGCGCCGAG-3’(配列番号9)及びプライマーRv:5’-TTACTTGTACAGCTCATC(配列番号10)を設計し、pAcGFP1(タカラバイオ)を鋳型として用いてPCRを行った。増幅断片とSspI消化したpUMGP16M13とのライゲーションを行い、得られたライゲーションサンプルを用いてpUM13GFPを取得した。 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.
Mms13のN末端にBAP、C末端に緑色蛍光タンパク質GFPを融合したタンパク質を発現するpUMBAPM13GFPを構築するため、NsiIサイト(下線を表示した配列)を付加したプライマーNsiI−BAP−F:5’-ATGCATATGGCTCAGCGTCTGTTCCA-3’(配列番号11)、及びNsiI−BAP−R:5’-ATGCATAGAACCACCTTTCGGACCGT-3’(配列番号12)を設計し、pUM13GFPBAPをテンプレートとしてPCRを行った。PCR産物をpCR4−Blunt−TOPOにサブクローニングし、インサート配列のシークエンス解析を行った。次いで、NsiIを用いてインサート配列を切り出し、NsiI消化したプラスミドpUM13GFPとのライゲーションを行った。得られたライゲーションサンプルを用いて大腸菌DH5αを形質転換した。得られたコロニーからプラスミドを抽出し、インサート配列の挿入方向をシークエンス解析により確認した(図2)。 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).
Mms13のC末端にBAP、mBCCP、及びmBCCP78を融合したタンパク質をそれぞれ発現するpUM13BAP、pUM13mBCCP、及びpUM13mBCCP78を構築するため、BAP増幅プライマーとしてBAP−F:5’-ATGGCTCAGCGTCTGTTCCA-3’(配列番号13)、BAP−R:5’-AGAACCACCTTTCGGACCGT-3’(配列番号14)を、mBCCP及びmBCCP78増幅プライマーとしてmBCCP−F:5’-ATGGGCAACAAGACTCCCATC-3’(配列番号15)、mBCCP78−F:5’-CATCCCGGCGCGGTG-3'(配列番号16)、mBCCP−R:5'-CTATTCGATGATCAGCAAGGGC-3'(配列番号17)を設計した。pUM13GFPBAPをテンプレートとしてBAPのPCR増幅をおこなった。また、磁性細菌Magnetospirillum magneticum AMB−1ゲノムをテンプレートとしてmBCCP及びmBCCP78のPCR増幅を行った。それぞれのPCR産物とSspI消化したプラスミドpUMGP16M13とのライゲーションを行い、得られたライゲーションサンプルを用いて大腸菌DH5αを形質転換した。得られたコロニーからプラスミドを抽出し、インサート配列の挿入方向をシークエンス解析により確認した(図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.磁性細菌の形質転換、培養及びBacMPsの調製
野生株の磁性細菌Magnetospillum magneticum AMB−1はMSGM(Blakemore et al. 1979)4.5lに植菌し、アルゴンガスを15分間バブリングすることにより微好気状態にした上で、室温にて約5日間、静置培養した。また、エレクトロポレーションによりプラスミドpUMBAPM13GFPを導入した形質転換体は5μg/ml、アンピシリン含有MSGMで培養し、pUM13mBCCP、及びpUM13mBCCP78を導入した形質転換体は5μg/mlアンピシリン、50μMビオチン含有MSGMで培養した。
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.
培養した菌体は7200rpm、4℃で10分間、遠心分離することにより集菌し、リン酸緩衝生理食塩水(PBS、pH7.4)30mlに懸濁した後に、フレンチプレスを用いて2000kg/cm2で破砕した。その後、菌体破砕液を入れた三角フラスコの底部にネオジウム−ボロン(Nd−B)磁石を取り付けて磁気微粒子(BacMPs)を磁気分離し、2−[4−Hydroxyethyl]−1−piperazinyl]ethanesulfonic acid(HEPES)緩衝液(10mM、pH7.4)中で超音波洗浄機を用いて10回洗浄した。洗浄したBMPsはPBSに懸濁し、4℃で保存した。 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.大腸菌由来ビオチンリガーゼによるBAPのビオチン標識
50mMのMgCl2、10μMビオチン、100mMのATP、及び10μg/ml大腸菌由来ビオチンリガーゼ含有PBS(pH7.4、100μl)中に50μgのBacMPsを懸濁し、室温で1時間撹拌した。その後、磁気分離したBacMPsを50μlのPBSで3回洗浄した。
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.ビオチン標識BacMPsへのストレプトアビジンの固定化
磁性細菌M. magneticum AMB-1形質転換体から抽出したBacMPs10μgに対し、20μg/mlのTRITC標識ストレプトアビジン(TRITC−SA)を100μl添加した、室温で1時間撹拌した。その後、100μlのPBSTで3回洗浄し、BacMPsを400μlのPBSTに懸濁し、蛍光顕微鏡で観察した。
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.
磁性細菌M. magneticum AMB-1形質転換体から抽出したBacMPs(50μg)に対し、1/100希釈アルカリフォスファターゼ標識ストレプトアビジン(ALP−SA)溶液(タンパク質濃度5.4mg/ml、2mg/mlのBSA含有)を100μl添加し、室温で1時間撹拌した。100μlのPBSTで3回洗浄した後、BacMPsを50μlのPBSに懸濁し、ルミホス530(3.3×10−4mol/l、50μl)を加え、5分後の発光強度を測定した。 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.
BacMPs上に固定化できるストレプトアビジン量を測定するため、TRITC標識ストレプトアビジンを固定化し、蛍光強度を測定した。200μlのTRITC標識ストレプトアビジン溶液を100μgのBacMPsに添加し、室温で1時間撹拌した。その後、200μlのPBSTで3回洗浄し、50μgのBacMPsを500μlのPBSに懸濁した。その後、粒子の蛍光強度(励起波長:545nm、蛍光波長:580nm)を、蛍光分光光度計を用いて測定した。検量線作成のため、50μgの野生株由来BaMPs(WT−BacMPs)に既知濃度のTRITC溶液(500μl)を添加し、同様に蛍光強度を測定した。 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.ドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動
磁性細菌Magnetospirillum magneticum AMB-1から抽出した2mgの磁性細菌粒子(BacMPs)を1%ドデシル硫酸ナトリウム(SDS)溶液に懸濁し、100℃で30分間煮沸することでBacMPs膜タンパク質を抽出した。抽出した膜タンパク質をSDSサンプルバッファー(終濃度:6.25 mM Tris-HCl [pH6.8], 5 % 2-mercaptoethanol, 2 % SDS, 5 % sucrose and 0.002 % bromophenol blue)に混合し、12.5 %(wt/vol)ポリアクリルアミドゲルを用いて電気泳動を行った。その後、クマシンブリリアントブルーR−250を用いてゲルを染色した。
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.ビオチン標識BacMPsへのストレプトアビジンの固定化
BacMPs上にストレプトアビジンを固定化できることを確認するため、金ナノ粒子標識ストレプトアビジンを固定化し、透過型電子顕微鏡による観察を行った。PBSTを用いて1/2希釈した200μlの金ナノ粒子標識ストレプトアビジン溶液を10μgのBacMPsに添加し、室温で1時間撹拌した。その後、100μlのPBSTで3回、100μlの超純水で1回洗浄した。洗浄後、BacMPsを超純水に懸濁し、グリッド上で30分間乾燥させた。その後、透過型電子顕微鏡(H700H、日立製作所)を用いて粒子を観察した。
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.結果
2−1.mBCCP(78)提示BacMPsのインビボ(in vivo)ビオチン標識
pUM13mBCCP形質転換体から磁気微粒子(mBCCP−BacMPs)を抽出し、TRITC−SAを用いてビオチン標識の有無を確認した。その結果、mBCCP−BacMPs上にTRITC−SAが固定化している様子が観察された。このことから、BacMPs上に提示されたmBCCPが磁性細菌体内においてビオチン標識されることが示された(図3)。
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).
次いでBacMPs上にディスプレイしたビオチン標識タグ、mBCCPAの短縮化を試みた。一般的にBCCPの持つアミノ酸配列のうち、ビオチン化に関与する配列はC末端側に集中していることが知られている。そこで、磁性細菌M. magneticum AMB−1由来mBCCPのアミノ酸配列を、近縁種であるM. magnetotacticum MS−1、M. gryphiswaldense MSR−1、Rhodospirirum rubrum由来のBCCPと比較した(図4)。その結果、72〜149残基目のC末端側78残基(mBCCP78)に相当する部分に共通配列が集中していることがわかった。そこで、mBCCP78をMms13のC末端に融合したタンパク質を発現するプラスミドpUM13mBCCP78を用いてM. magneticum AMB−1を形質転換し、磁気微粒子(mBCCP78−BacMPs)を抽出した。 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.
このmBCCP78−BacMPsが磁性細菌体内においてビオチン標識されているかをSA−ALPを用いて確認しところ、mBCCP−BacMPsと同程度の発光強度を示した(図5)。このことから、149アミノ酸残基からなるmBCCPのうち、C末端側78アミノ酸残基のみを持つmBCCP78も、磁性細菌体内においてビオチンリガーゼによって認識されてビオチン標識されることが示された。 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.BAP提示BacMPsのインビトロ(in vitro)ビオチン標識
大腸菌由来ビオチンリガーゼの働きによりBacMPs上に発現したBAPがビオチン標識されることを確認するため、ビオチンリガーゼ処理もしくは未処理のBAP−Mm13−GFP発現BacMPsとMm13−GFP発現BacMPsにSA−ALPを添加した。その結果、ビオチンリガ−ゼ処理したBAP−Mm13−GFP発現BacMPsにおいてのみ高い発光強度が得られた(図6)。このことから、ビオチンリガーゼを用いてBacMPs上のBAPを特異的にin vitroビオチン標識できることが示された。また、ビオチンリガーゼ未処理BAP−Mm13−GFP発現BacMPsにALP−SAが結合しなかったことから、BAPはAMB−1体内ではビオチン標識されないことが示された。
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.
なお、図5及び図6に示したグラフの縦軸は、ストレプトアビジン標識したアルカリフォスファターゼの活性を蛍光強度により表したものである。用いた磁気微粒子(BacMPs)の量が同じにもかかわらず、蛍光強度が約10倍異なることからインビトロでのビオチン化反応に比べて、磁性細菌内におけるビオチン化反応の方が顕著に効率的であることが分かる。 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.BacMPs上におけるMms13−mBCCP融合タンパク質の発現確認
M. magneticum AMB-1野生株、およびBacMPs上の膜タンパク質Mms13とAMB−1由来ビオチン結合タンパク質mBCCPとの融合タンパク質を発現するプラスミドpUM13mBCCP形質転換体から得られたBacMPs上の膜タンパク質を抽出し、SDS−PAGEを行った。その結果を図7に示す。pUM13mBCCP形質転換体から得られたBacMPs(mBCCP−BacMPs)において、30から43kDa付近に顕著なバンドが得られた。Mms13−mBCCP融合タンパク質は約31kDaのタンパク質であることから、SDS−PAGEにより得られたバンドはMms13−mBCCPであると考えられた。以上の結果より、Mms13−mBCCP融合タンパク質がBacMPs上に高発現に導入されていることが確認された。
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.BacMPs上に固定化された金ナノ粒子の観察
BacMPs上にストレプトアビジンが導入されていることを確認するため、WT−BacMPsおよびmBCCP−BacMPsに金ナノ粒子標識ストレプトアビジンを添加し、透過型電子顕微鏡で観察した。その結果を図8に示す。mBCCP−BacMPs上にはWT−BacMPsに比べて多くの金ナノ粒子が固定化されていることが確認された。このことから、BacMPs上に提示されたmBCCPがビオチン化され、そこにストレプトアビジンを固定化できることが示された。
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.BacMPs上に固定化されたTRITC標識ストレプトアビジン量の測定
BacMPs上に固定化できるストレプトアビジン量を測定するため、WT−BacMPsおよびmBCCP−BacMPsにTRITC標識ストレプトアビジンを添加し、蛍光強度を測定した。その結果を図9に示す。1mgのmBCCP−BacMPs上に約1300ngのストレプトアビジンを固定化できることが示された。これは一粒子のBacMPs(粒径:75nm、密度:5.2g/cm3と近似)上に約15分子のストレプトアビジン(60kDa)を固定化できることを示している。
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 ).
Claims (11)
前記磁性細菌から磁気微粒子を単離する工程と、
を含むことを特徴とするビオチン標識磁気微粒子の製造方法。 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:
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