JP4047482B2 - Substance measurement sensor - Google Patents

Description

【００４９】
図16は図15で示された結果の内、それぞれ 1xCamR-MalE, 2 x CamR-MalE, および3xCamR-MalE融合タンパク質のDNAに対する結合実験の結果の典型的な例を抜き出し、一定時間のレスポンスカーブを一枚のグラフに重ね合わせて示したものである。
【００５０】
図17は図15で示された結果の内、それぞれ 1 x CamR-MalE, 2 x CamR-MalE, および3 x CamR-MalE 融合タンパク質のDNAに対する結合実験で、ある時間の共鳴角の3回の結果を平均化して示したものである。
【００５１】
図18はアニーリングさせたDNAを100 pmol 用いてセンサーチップに固定化させ、それぞれ 5 mMのアミロースレジンカラムを用いて精製した2 x CamR-MalE 融合タンパク質または融合タンパク質をfactor Xa で消化後、DEAEイオン交換カラムクロマトグラフィーを用いて分離精製した5 mMの2 x CamRタンパク質を用いてSPR実験を行った場合の共鳴角の時間的変化を示す。同じ試料を用いて3回繰り返し実験を行っている。一枚のセンサーチップを用いた連続実験である。なお、図18中の各矢印で示される数字における操作の内容は下記表２に示す通りである。
【００５２】
【表２】

【００５３】
図19は図18で示された結果の内、 2 x CamR-MalE 融合タンパク質, および2 x CamRのDNAに対する結合実験で、ある時間の共鳴角の3回の結果を平均化して示したものである。
【００５４】
図20は濃度の違う5 mMおよび10 mMの2 x CamR-MalE 融合タンパク質のDNAに対する結合実験で、ある時間の共鳴角の3回の結果を平均化して示したものである。
【００５５】
図21は図18で示された結果の内、精製した2 x CamRタンパク質を用いて100 fg/ml, 1 ng/ml, 10 μg/mlの濃度のカンファーを用いてカンファーの定量性調べた実験の結果の典型的な例を抜き出し、一定時間のレスポンスカーブを一枚のグラフに重ね合わせて示したものである。
【００５６】
図22は図18で示された結果の内、精製した2 x CamRタンパク質を用いて100 fg/ml, 1 ng/ml, 10 μg/mlの濃度のカンファーを用いてカンファーの定量性を調べた実験で、ある時間の共鳴角の3 回の結果を平均化して示したものである。
【００５７】
センサーに用いるCamRタンパク質は構築した大量発現用プラスミドを保有する組換え体（XL1-blue+1xCamR、XL1-blue＋2xCamR、XL1-blue+3xCamR）を培養して、マルトース結合タンパク質の融合タンパク質として大量発現させる系によって、容易に精製することができた。CamRタンパク質は本精製法によって際限なく得ることが可能である。また精製したCamRタンパク質は- 20 ℃または- 85 ℃で長期間保存可能であり、4 ℃でも２週間以上安定である。
【００５８】
図18に示されるように、SPRによるカンファーの測定はリアルタイムにデータを得ることができ、これに要した時間はDNAの固定から検出まで、一つのサンプルを20分未満で終了することができた。センサーチップはglycine-HCl (pH 1.0)溶液を用いて再生することができ、一枚のチップで10 回以上繰り返し使用することができた。また、センサーチップは超音波洗浄することによって再利用することができた。
【００５９】
同じサンプルを繰り返し行った場合、ほぼ同じ共鳴角が得られ、再現性が得られた（図15および18）。カンファーを加えないCamRタンパク質のみのコントロールの共鳴角はカンファーを添加したもの (1 ng/ml および10μg/ml )より大きな値を示した（図21および図22）。この結果より、本実施例の原理で予測したように、カンファーに結合したCamRはカンファーの濃度依存的にDNAの結合が阻害されることが示された。
【００６０】
図19よりSPRを用いて調べたCamRタンパク質のDNAの結合性は、CamRタンパク質がMalEタンパク質との融合タンパク質の状態より分離されたCamRタンパク質のみを用いた方が強いことが知られた。これはCamRタンパク質のMalEタンパク質との融合タンパク質の状態ではMalEタンパク質によって、DNAに結合するためのコンフォメーションを取ることが阻害されるためであると考えられる。このことからSPRに供するCamRタンパク質はfactor Xaで切断した後に、DEAEイオン交換カラムクロマトグラフィーを用いて分離精製したものを用いた方が、臭気物質の検出感度の向上が見込まれた。
【００６１】
図20より5 mMと10 mMの2xCamR-MalE 融合タンパク質の共鳴角を比較すると、5 mMのものより10 mMのものの方が共鳴角が高く、CamRタンパク質の濃度が高いほど共鳴角が高くなる傾向が見られた。これはSPRを用いた分子間の相互作用を分析する反応において結合する物質濃度が高いほどレスポンスが大きくなる事実と一致する。
【００６２】
図21および図22より臭気物質カンファーは10 pg/ml〜100 ngの間の濃度を定量検出することができた。検出限界は100 fg/ml〜10 pg/mlの間であるが、正確な値は得られていない。この感度のレベルはヒトが鼻で感じることができるよりも10〜100倍高く、ガスクロマトグラフ質量分析計を用いた場合と同等またはそれ以上である。
【００６３】
本実施例に用いている材料のDNAおよびCamRタンパク質は物質であり、取り扱いや実験場所に制限を受けない。感度はcamオペロンを微生物の発光遺伝子オペロンを用いた系の場合が1 ng / mlの検出限度（開発者らの以前のデータ）であったものが、本研究のSPR方法の場合が10 pg / mlである。従って、検出感度は本研究のSPRを用いた方法の方が、発光遺伝子を用いた方法より100 倍高い。測定時間は発光遺伝子を用いた場合20 時間要したものが、本研究のSPRを用いた方法は20分未満であり、大幅に測定時間を短縮することができる。
【００６４】
実施例２
（Ａ）(1) CamR の大量生産用プラスミドの構築
１) His-tagged CamRの構築（図２３）
PCRに用いるためのHtnt-2プライマー5'-ATGCATCACCATCACCATCACATGGACATCAAG-3'はT4 ポリヌクレオチドキナーゼ処理してリン酸化した。1回目のPCRはpLC87を鋳型にして、Htnt-1プライマー5'-ATCACCATCACATGGACATCAAGCAGAGCTTGCTG-3'とr7プライマー5'-GAGGAACACCAGATGTCGTT-3'を用いて10サイクル行った。2回目のPCRは1回目のPCR反応液1μlを鋳型にして、リン酸化したHtnt-2プライマーとr7プライマー5'-GAGGAACACCAGATGTCGTT-3'を用いて25サイクル行った。増幅した断片はEco RIで切断した上で、ポリアクリルアミドゲル電気泳動し、1058 bpの断片を分離回収した。この断片をpMal-c2のXmn IとEco RIサイトに挿入した。得られたプラスミドをpHis-tagged camRとして、His-tagged CamRの大量発現用プラスミドとして用いた。
【００６５】
(2) CamR の大量生産用プラスミドを保有する大腸菌からの CamR の精製
１） マルトース結合タンパク質（MalE)とCamRタンパク質の融合タンパク質の精製
CamR大量生産用プラスミドは大腸菌XL1-blueに導入した。得られた組換え体はDH5α+His-tagged CamRと名付け、以下、実施例１と同様にしてFactor XaによりMalEとCamRとに切断した。
【００６６】
２) Ni-NTAカラムクロマト法による精製法
1 mg/ml のタンパク質溶液 1 ml は1.5 ml 容のマイクロチューブに移し、４μgのFactor Xa (New England biolabs, Inc)を加え16 ℃で一晩インキュベートして、消化した。消化した10 mgのタンパク質はCentriplus 10 (Amicon,Inc.)を用いて溶解バッファー (50 mM NaH₂PO₄ pH8.0, 300 mM NaCl, 10 mMイミダゾール) に交換した。5 mlのNi-NTA Superflow レジン (QIAGEN)を1x10 cm のカラムに注いだ。カラムを40 mlの溶解バッファーで平衡化させた。溶解バッファーに交換したタンパク質を0.5 ml／minの流速でカラムに加えた。カラムを40 mlの溶解バッファーで洗った。カラムを40 mlの洗浄バッファー (50 mM NaH₂PO₄, pH8.0, 300 mM NaCl, 20 mMイミダゾール) で洗った。カラムに40 mlの溶出バッファー (50 mM NaH₂PO₄, pH8.0, 300 mM NaCl, 250 mMイミダゾール)を加えHis-tagged CamRを溶出させた。1 mlずつフラクションを回収した。回収したタンパク質はCentriplus 10 (Amicon,Inc.)を用いてカラムバッファー ( 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 10 mM β-メルカプトエタノール)に交換するとともに濃縮した。タンパク質はプロテインアッセイキットI (BIO-RAD Laboratories)を用いて定量し、その後0.5 ml容マイクロチューブに分注し、SPR実験に使用するまで - 85℃保存した。
【００６７】
(3) カンファーによる CamR の解離実験
実施例１と同様にして、DNAの固定→buffer交換→BSAによるブロッキング→グリシンHCl (pH1.0)によるフリーのBSAの洗い→CamR結合バッファーによる平衡化の一連の操作を行った。その後、10μlの10 mM CamR溶液を加えた。安定したところでエタノールに溶解した20 mMカンファー溶液をカラムバッファーで希釈した1pg/ml〜100ng/mlまでの濃度のカンファーを2μl 加えた。コントロールとして2 μlのカラムバッファーを添加した試料を用いた。この実験は２回行った。
【００６８】
(4) CamR 二量体形成のモニタリング実験
実施例１と同様にして、DNAの固定→buffer交換→BSAによるブロッキング→グリシンHCl (pH1.0)によるフリーのBSAの洗い→CamR結合バッファーによる平衡化の一連の操作を行った。その後、10μlの10 mM CamR溶液を加え、20 分以上静置した。
【００６９】
(5) かび臭物質 2-MIB の定量
カンファーと同様に上水におけるかび臭物質 2-メチルイソボルネオール(2-MIB)の定量性や検出限界を求める実験を行った。2-MIBはエタノールに溶解して20 mg/mlの濃度の保存液を調製した。この保存液は蒸留水で希釈し1 pg/ml〜10μg/mlの濃度に調製した。実験にはこの2-MIBの希釈液と10 mM His-tagged CamRの混合液10 μlを用いた。
【００７０】
（Ｂ） 実験結果
１) 構築したプラスミドのマルトース結合タンパク質とCamRタンパク質をコードする遺伝子領域を含む塩基配列を図２４，２５に示す。
【００７１】
２）MalE-CamR融合タンパク質からのCamRの精製は、CamRのアミノ末端に6 残基のヒスチジンを付加して発現したタンパク質をNi-NTAカラムによって分離精製する方法によって容易に高純度のタンパク質を大量に分離精製することができた。本方法はイオン交換カラムを用いて精製する方法と較べ、MalEやFactor Xaによって切れ残った融合タンパク質の混入もないため純度が高く、回収率も高かった。また、アミノ末端にヒスチジンが付加された融合タンパク質はFactor Xaによる消化が著しく高まり、少ない酵素量（以前の1/5量）で切れ残りがほとんどなくなった。このため、本方法によるCamRの生産および精製法は高価なFactor Xa の使用量が少なくて済むためコストを低くすることができることも有益である。
【００７２】
３） カンファーによるDNAからCamRの解離度を調べる実験は図２６に示す共鳴角の減少量の値を用いた。この結果、1 pg/ml 〜 100 ng/mlの濃度のカンファーを添加した場合の共鳴角の減少度はbufferのみのコントロールの減少度と同程度かこれより低く、著しく大きくなることはなかった。このため、カンファー添加によるDNAからCamRの解離現象を検出することはできなかった。従って、臭気物質によってDNAからCamRが解離する現象をもとに臭気物質量を測定する方法は適用できないことが示された（図２７、２８）。なお、図２７中の各矢印で示される数字における操作の内容は下記表３に示す通りである。
【００７３】
【表３】

【００７４】
４）CamRタンパク質のDNAの結合性は予めCamRタンパク質とカンファーまたは2-MIBを結合させた混合試料をDNAに結合させたところ、カンファーまたは2-MIBの添加量に応じた結合が示された（図３１）。また、この場合の共鳴角の変化量はカンファーまたは2-MIBの濃度の違いを十分に捕らえることができた。従って、かび臭物質の検出にはCamRタンパク質のかび臭物質によるDNAへの結合阻害度から求める方法が適している。本方法によってSPRを応用したかび臭バイオセンサーの基本原理が完成した。
【００７５】
５）実施例１又は実施例２で作製した、MalE-1xCamR融合タンパク質、His tagged CamR、MalE-2xCamR融合タンパク質、2xCamRタンパク質、MalE-3xCamR融合タンパク質のDNAの結合性はHis-tagged CamRが圧倒的に高いDNA結合を示した（図２９）。ただし、コントロールはCamRを加えないバッファーのみの試料であり、また濃度は2xCamRタンパク質が5 mMであり、他は10 mMの濃度のタンパク質である。また、値は３回の平均値である。この結果はMalEまたはタンデムに繋がった際のCamRの構造がDNA結合を阻害しているためであると考えられ、そのため以後の実験にはNi-NTAカラムによって精製した単独のCamRタンパク質を用いた。
【００７６】
６）CamRタンパク質のDNA結合状態の時間変化をSPRでモニターした所、約10 分までは一つの右上がりのDNA結合曲線が検出されたが、その後にもう一つの右上がりの曲線が検出された（図３０）。Aramakiら（上掲)は、CamRがDNAに結合する際、二量体で結合すると報告している。この考察から推測すると、最初の反応は1つ目のCamRがDNAに結合した反応であり、次の反応は、2つ目のCamRが1つ目のCamRとDNAに結合した反応であるかも知れない。すなわち、CamRのDNAの結合はCamRが二量体となってからDNAに結合するのではなく、一つのCamRが結合した後に、DNAの上でもう一つのCamRが結合すると考えられる。本研究の臭気物質の測定には一つ目の反応を用いている。しかしながら、今後、2つ目の反応を用いて臭気物質を測定すれば、トータルの反応は大きくなり、感度の向上に役立つかも知れない。
【００７７】
７）上水におけるかび臭物質2-メチルイソボルネオール(2-MIB)を本センサーによって測定したところ、1 ng／L〜10 mg／Lまで定量的に検出することができた（図３１）。このレベルはヒトの鼻の約数十倍であり、ガスクロマトグラフ質量分析計(GC-MS)法と同程度である。
【００７８】
【発明の効果】
以上のように、本発明により、微生物に比較して取り扱いが簡便で、培養が不要で測定に要する時間が短く、かつ、従来の微生物センサーよりも高感度で臭気物質等の物質を測定することができる物質測定用センサーが提供された。
【００７９】
【配列表】

【００８３】

【００８４】

【００８５】

【００８６】

【００８７】

【００８８】

【００８９】

【００９０】

【００９１】

【００９２】

【００９３】

【００９４】

【００９５】

【図面の簡単な説明】
【図１】本発明のセンサーを用いた測定方法の原理を説明するための模式図である。
【図２】本発明の実施例で作製した、ｃａｍＲタンパク質の単量体とＭａｌＥタンパク質との融合タンパク質を発現するベクターであるp1xcamRを構築する方法を説明するための図である。
【図３】本発明の実施例で作製した、ｃａｍＲタンパク質の二量体とＭａｌＥタンパク質との融合タンパク質を発現するベクターであるp2xcamRを構築する方法を説明するための図である。
【図４】図３の続きを示す図である。
【図５】図４の続きを示す図である。
【図６】本発明の実施例で作製した、ｃａｍＲタンパク質の三量体とＭａｌＥタンパク質との融合タンパク質発現するベクターであるp3xcamRを構築する方法を説明するための図である。
【図７】図６の続きを示す図である。
【図８】 p1xcamRベクター中の、ｃａｍＲタンパク質の単量体とＭａｌＥタンパク質との融合タンパク質をコードする領域及びその近傍の塩基配列を、それがコードするアミノ酸配列と共に示す図である。
【図９】図８の続きを示す図である。
【図１０】 p2xcamRベクター中の、ｃａｍＲタンパク質の二量体とＭａｌＥタンパク質との融合タンパク質をコードする領域及びその近傍の塩基配列を、それがコードするアミノ酸配列と共に示す図である。
【図１１】図１０の続きを示す図である。
【図１２】 p3xcamRベクター中の、ｃａｍＲタンパク質の三量体とＭａｌＥタンパク質との融合タンパク質をコードする領域及びその近傍の塩基配列を、それがコードするアミノ酸配列と共に示す図である。
【図１３】図１２の続きを示す図である。
【図１４】図１３の続きを示す図である。
【図１５】実施例１で行なった、ＳＰＲ実験における時間と共鳴角との関係を示す図である。
【図１６】図15で示された結果の内、それぞれ同濃度の1xCamR-MalE, 2 x CamR-MalE, および3xCamR-MalE融合タンパク質のDNAに対する結合実験の結果の典型的な例を抜き出し、一定時間のレスポンスカーブを一枚のグラフに重ね合わせて示した図である。
【図１７】図15で示された結果の内、それぞれ 1 x CamR-MalE, 2 x CamR-MalE, および3 x CamR-MalE 融合タンパク質のDNAに対する結合実験で、ある時間の共鳴角の3回の結果を平均化して示した図である。コントロールはタンパク質の溶解に用いたバッファーのみを添加したものである。
【図１８】本発明の実施例において、各種濃度のカンファーを測定したＳＰＲ実験における時間と共鳴角との関係を示す図である。
【図１９】図18で示された結果の内、2 x CamR-MalE 融合タンパク質, および2 x CamRのDNAに対する結合実験で、ある時間の共鳴角の3回の結果を平均化して示した図である。
【図２０】本発明の実施例において、濃度の違う5 mMおよび10 mMの2 x CamR-MalE 融合タンパク質のDNAに対する結合実験で、ある時間の共鳴角の3回の結果を平均化して示した図である。
【図２１】図18で示された結果の内、精製した2 x CamRタンパク質を用いて100 fg/ml, 1 ng/ml, 10 μg/mlの濃度のカンファーを用いてカンファーの定量性調べた実験の結果の典型的な例を抜き出し、一定時間のレスポンスカーブを一枚のグラフに重ね合わせて示した図である。
【図２２】図18で示された結果の内、精製した2 x CamRタンパク質を用いて100 fg/ml, 1 ng/ml, 10 μg/mlの濃度のカンファーを用いてカンファーの定量性を調べた実験で、ある時間の共鳴角の３回の結果を平均化して示した図である。
【図２３】本発明の実施例で作製した、ヒスチジンが６個連続して成るタグを含むｃａｍＲタンパク質の単量体とＭａｌＥタンパク質との融合タンパク質を発現するベクターであるpHis-tagged CamRを構築する方法を説明するための図である。
【図２４】 pHis-tagged CamRベクター中の、ｃａｍＲタンパク質の単量体とＭａｌＥタンパク質との融合タンパク質をコードする領域及びその近傍の塩基配列を、それがコードするアミノ酸配列と共に示す図である。
【図２５】図２４の続きを示す図である。
【図２６】実施例２において、試料液にカンファーを加える前後の共鳴角の変化を示す図である。
【図２７】実施例２で行なったＳＰＲ実験における時間と共鳴角との関係を示す図である。
【図２８】実施例２で行なったＳＰＲ実験における、添加したカンファー濃度と共鳴角との関係を示す図である。
【図２９】本発明の実施例で作製した、各種リプレッサータンパク質とcamオペロンＤＮＡとの結合性を示す図である。
【図３０】実施例２において行った、CamR二量体形成のモニタリング実験における時間と共鳴角との関係を示す図である。
【図３１】実施例２において行った、２−メチルイソボルネオールの測定実験により得られた、２−メチルイソボルネオールの濃度と共鳴角との関係を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sensor for measuring various substances such as odor substances.
[0002]
[Prior art]
As an odor sensor for detecting an odor such as a mold odor substance, a microbial sensor produced by a genetic engineering technique is known (Japanese Patent Laid-Open No. Hei 8-242857). This microbial sensor includes an operator / promoter region in a camphor hydroxylase operon (cam operon) and a reporter gene that is inserted downstream of the region and is controlled by the region, and the expression of which can be detected. For example, a microorganism transformed with a recombinant vector containing a β-galactosidase gene or a luciferase gene group. The cam operon is an operon derived from the CAM plasmid of Pseudomonas keptida that can grow using camphor (camphor) as the sole carbon source, and acts at the initial stage of camphor degradation. When an odorous substance such as camphor is present in the test sample, the odorous substance acts on the operator / promoter region and a downstream reporter gene is expressed, so that the odorous substance can be detected.
[0003]
[Problems to be solved by the invention]
The known microbial sensor has advantages such as high specificity with respect to a recognition substance and an increase in the number of materials that are microorganisms without limit, so that the material cost is low. However, the microbial sensor is inconvenient to handle because the sensor itself is a microorganism, and has a problem that it takes a measurement time because it requires a culture time. Needless to say, it is more preferable if the detection sensitivity is further improved.
[0004]
Therefore, the object of the present invention is to measure substances such as odorous substances with a higher degree of sensitivity than conventional microorganism sensors, which is easier to handle than microorganisms, does not require culturing, has a short measurement time, and is more sensitive than conventional microorganism sensors. It is to provide a sensor for measuring substances.
[0005]
[Means for Solving the Problems]
As a result of earnest research, the inventor of the present application has achieved a simple and short by using a sensor formed by binding a nucleic acid that binds to a repressor protein that binds to a substance to be measured on the surface of a sensor chip of a surface plasmon resonance apparatus. The inventors have found that substances can be measured with high sensitivity over time, and the present invention has been completed.
[0006]
That is, the present invention is a substance obtained by binding a nucleic acid that binds to a repressor protein that binds to a substance to be measured on the surface of the sensor chip of the surface plasmon resonance apparatus opposite to the side on which light is incident during measurement. Provide a sensor for measurement.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The surface plasmon resonance (SPR) method is performed when the refractive index of a medium such as a solution in contact with the lower surface of a sensor chip (a metal substrate such as gold deposited on a glass substrate) changes. This is a method for measuring the change in the refractive index of a medium by utilizing the change in the angle of the reflected light with reduced intensity among the reflected light incident at various angles through the prism (Seiichi Kasai, Protein Nucleic acid enzyme, Vol.37 No.15 (1992), pp.2977-2984). The intensity of the reflected light reflected at a specific angle decreases because resonance occurs when the wave number of the surface wave called surface plasmon generated on the surface of the metal thin film coincides with the evanescent wave generated at the interface between the glass and the metal thin film, This occurs because part of the energy of the light is used to excite surface plasmons and reduces the amount returned as reflected light. If one of the reactants involved in the chemical reaction is bound to the lower surface of the sensor chip, the refractive index of the medium that contacts the lower surface of the sensor chip changes as the reactant reacts with the other reactant, The angle of the reflected light with reduced intensity (this angle is called “resonance angle”) changes from moment to moment as the chemical reaction proceeds. Therefore, the progress of the chemical reaction can be observed in real time by measuring the change in the resonance angle in real time. For this reason, SPR is used for the study of chemical reaction kinetics, and in the field of biochemistry, it is used for analysis of antigen-antibody reaction.
[0008]
The sensor of the present invention utilizes the above SPR device. Since the SPR device itself is commercially available, a commercially available product can be used. In the sensor of the present invention, a nucleic acid that binds to a repressor protein that binds to a substance to be measured is bound on the lower surface of the sensor chip of the SPR device (that is, on the opposite side of the light incident side during measurement). Yes. As the nucleic acid to be bound, any nucleic acid can be used as long as it binds to the repressor protein that binds to the substance to be measured. A preferred example of such a nucleic acid is an operator sequence in an operon including a gene encoding the repressor protein. An example is an operator in the cam operon that binds to a camphor repressor protein. The nucleotide sequence of the operator in the cam operon is known, and Aramaki, H., et al., (1993) J. Bacteriol., 175, 7828-7833, and Fujita, M. et al., (1993) J. Bacteriol, 175, 6953-6958. Repressor proteins encoded by the camR gene in the cam operon bind to the operator in the cam operon. Furthermore, if the operon of microorganisms in the environment is metabolized, in addition to organic substances that exist naturally, those that are artificially created by humans and released to the environment, benzene, xylene, toluene, naphthalene, phenol Since microbial operons containing genes encoding repressor proteins that bind to harmful organic substances such as organic heavy metals such as methylmercury are already known, operator sequences in these operons can also be used. A nucleic acid having a sequence in which one or a plurality of nucleotides are deleted, substituted, inserted or added in the operator sequence and which binds to the repressor protein can also be used. In particular, in order for a repressor protein to bind, a complete operator sequence is not required, and only a part of the operator sequence needs to be present. Therefore, such a partial sequence of an operator can also be preferably used. For example, as an example of a preferable operator partial sequence of the cam operon, there can be mentioned the base sequence shown in SEQ ID NO: 1 in the sequence listing. Needless to say, a nucleic acid having a sequence in which one or a plurality of nucleotides are deleted, substituted, inserted or added in the sequence shown in SEQ ID NO: 1 and which binds to a camphor repressor protein is also preferably used. be able to.
[0009]
Nucleic acid binding to the sensor chip surface of the SPR device is based on the SA membrane method using disulfide compounds (Taizo UDA et al., (1995) DENKI KAGAKU, 63, NO.12, 1160-1166), thiol membrane method, L -B membrane method (Ryuichi Yasuda et al. (1997) IEICE Transactions C-II Vol. J80-C-II No.1 pp.1-7) can be used by a method known per se. Among these, the SA membrane method is preferable because nucleic acid can be immobilized easily and the sensor chip can be reused. By these methods, one end of the nucleic acid can be bound to the surface of the sensor chip, and no part other than the end of the nucleic acid is in contact with anything, so the nucleic acid immobilized on the surface of the sensor chip is not repressor. It can freely bind to proteins. A specific example of a method for bonding DNA having the sequence shown in SEQ ID NO: 1 to a sensor chip deposited with gold on a glass substrate by the SA film method is described in detail in the following examples. The amount of nucleic acid to be bound to the surface of the sensor chip is not particularly limited and may be any amount as long as the substance in the sample can be measured, but is usually 1 mm.²It is about 500 to 2000 pmol per unit.
[0010]
In the measurement, a sample that may contain a substance to be measured added with the repressor protein is brought into contact with the lower surface of the sensor chip, and light at various angles is placed on the upper surface of the sensor chip. The resonance angle is measured in real time by measuring the intensity of the reflected light. If the sample contains a substance to be measured, the substance binds to the repressor protein, so the repressor protein cannot bind to the nucleic acid immobilized on the sensor chip. Hardly changes before and after the measurement. On the other hand, when the substance to be measured is not contained in the sample, the repressor protein binds to the nucleic acid, so that the resonance angle is greatly increased. Since the degree of increase of the resonance angle depends on the amount of the substance contained, measuring the increase of the resonance angle enables not only detection of the substance in the sample but also quantification. Therefore, in this specification, “measurement” includes both detection and quantification. Note that the operation of making light incident on the sensor chip and the operation of measuring reflected light can be easily performed according to the instruction manual of a commercially available SPR device. FIG. 1 schematically shows the above measurement principle in the case of measuring camphor, which is an odorous substance, using camR protein as a repressor protein.
[0011]
In addition, the final concentration of the repressor protein added to the sample is not particularly limited and can be appropriately selected in light of the expected range of the amount of the substance to be measured. Usually, about 1 to 1000 mM is preferable, More preferably, it is about 5 to 200 mM. In addition, the repressor protein itself can be prepared in a large amount by a genetic engineering technique because the gene encoding it can be cloned by a known method (see Examples below).
[0012]
The repressor protein may be any one that can bind to the substance to be measured and the nucleic acid, and is not necessarily the same as the natural repressor protein. That is, one or more amino acids in the amino acid sequence of a natural repressor protein are substituted, deleted, or one or more amino acids are inserted or added, and the substance to be measured and the above Those capable of binding to a nucleic acid are also encompassed by the “repressor protein” referred to in the present invention. Accordingly, a fusion protein of a natural repressor protein and another protein that can bind to the substance to be measured and the nucleic acid is also included in the “repressor protein” referred to in the present invention.
[0013]
As such a modified repressor protein, for example, in the following examples, a protein in which a 6-residue histidine tag is added to the amino terminus of CamR is used. Included in “pressor protein”. By adding multiple histidine residues to the amino terminus of the repressor protein, high purity can be easily achieved by separation and purification using Ni-NTA (Ni-Nitrilotriacetic acid (Ni-NTA)) column. In this method, the repressor protein is first expressed as a fusion protein with other proteins, and then the repressor protein is purified by cleaving the fusion portion with Factor Xa or the like. Compared with the method of purification using an ion exchange column, there is no contamination with other proteins to be fused or uncut fusion protein, resulting in higher purity and higher recovery, and addition of histidine at the amino terminus. The digested fusion protein is significantly digested by Factor Xa, and there is almost no uncut residue with a small amount of enzyme (previous 1/5) For this reason, by adding multiple histidine residues to the amino terminus of the repressor protein and purifying with a Ni-NTA column, the amount of expensive Factor Xa used can be reduced, thereby reducing the cost. In this case, the number of histidine residues to be added is preferably about 4 to 8.
[0014]
In the case where the operator of the cam operon or a partial sequence thereof is used as the nucleic acid and the camR protein is used as the repressor protein, the binding between the operator or the partial sequence thereof and camR is smooth when the measurement is performed in the presence of calcium ions. This is preferable. In this case, the concentration of calcium ions is not particularly limited, but is preferably about 1 to 10 mM.
[0015]
The substance measured using the sensor of the present invention varies depending on the type of nucleic acid bound to the sensor chip and the type of repressor protein. For example, as the nucleic acid bound to the sensor chip, the operator sequence of the cam operon or its When a partial sequence is used and a camphor repressor protein is used as a repressor protein, mold odor substances such as camphor, 2-methyl-isoborneol and isoborneol can be exemplified. In addition, as described above, if the operon of microorganisms in the environment is metabolized, in addition to naturally occurring organic substances, humans artificially created and released to the environment, benzene, xylene, It can also be applied to the measurement of harmful organic substances such as toluene, naphthalene and phenol, and organic heavy metals such as methylmercury. The test sample may be any sample for which the substance contained therein is to be measured, for example, drinking water such as tap water, water such as the ocean, rivers, ponds, etc. Substances such as contained odor substances can be measured.
[0016]
The odor substance that reacts with the CamR protein belongs to a compound called monoterpene, including camphor. Monoterpenes are also known as scents for the majority of plants and fruits and as pheromones for some species of insects. CamR protein reacts not only with camphor but also with many bicyclic monoterpenes having a similar structure (JP-A-8-242857). In addition, the type of monoterpene that reacts with several types of CamR mutants varies depending on the mutant type. Some mutants have selectivity and react only with camphor (Japanese Patent Laid-Open No. 10-52276), react with not only bicyclic monoterpenes but also mono- and ring-opening monoterpenes (Japanese Patent Laid-Open No. 10-28591). By using these mutant CamR proteins in this method as in the wild type, in addition to the analysis of camphor, it can also be applied as a sensor to analyze the scent of plants (including food).
[0017]
The wild-type CamR protein also reacts to 2-methylisoborneol, a musty odorant in the waterworks, much like the structure of camphor. Some of the mutants react with geosmin, another musty odorous substance belonging to sesquiterpenes (JP-A-10-28591). Therefore, it is considered that the sensor of the present invention can be used for a water quality test for analyzing musty odor substances.
[0018]
【Example】
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples. In the following examples, the methods described in Sambrook, J. et al., (1989) Molecular Cloning 2nd Edition. Cold Spring Harbor Laboratory Press were used unless otherwise specified.
[0019]
Example 1
(A) (1)CamR Of plasmid for mass production
The construction of a plasmid for mass production of CamR uses the E. coli expression vector pMal-c2 (New England Biolabs, INC.), Which is expressed as a maltose binding protein-sample fusion protein and affinity purified using an amylose resin column. It was. However, the purification of CamR from the MalE-CamR fusion protein was performed by one of the following two methods. One is a method by ion exchange column chromatography (Example 1), and the other is a 6-residue histidine tag added to the amino terminus of CamR (introduced by PCR), and this His-tagged CamR is converted into Ni-nitrilo. A method (Example 2) for purification by column chromatography using triacetic acid (Ni-Nitrilotriacetic acid (Ni-NTA)) was employed.
[0020]
In the surface plasmon resonance reaction, the greater the molecular weight of the molecule to be reacted, the greater the resonance angle and the higher the sensitivity. CamR also binds in dimers when bound to DNA (Aramaki, H. supra).
[0021]
Taking these SPR principles and CamR properties into consideration, each camR gene linked to a maltose-linked gene is linked to one (1xcamR), two linked (2xcamR) or three (3xcamR) tandems. Thus, it was constructed to increase the SPR reaction by increasing the molecular weight. Four amino acid (glycine) residues were introduced between the camR genes by PCR.
[0022]
1) Construction of 1xcamR (Figure 2)
The camR-Nt primer 5′-ATGGACATCAAGCAGAGCTTG-3 ′ for use in PCR was treated with T4 polynucleotide kinase to phosphorylate the 5′-OH group. PCR was performed using phosphorylated camR-Nt primer and r7 primer 5′-GAGGAACACCAGATGTCGTT-3 ′ using pLC87 (JP-A-8-242857, FERM P-14818) as a template. The amplified fragment was cleaved with Eco RI and then subjected to agarose gel electrophoresis, and a 1037 bp fragment was separated and recovered. This fragment was inserted into the Xmn I and Eco RI sites of pMal-C2. The obtained plasmid was used as p1xcamR as a plasmid for mass expression of 1xCamR.
[0023]
2) Construction of 2xcamR (Figs. 3-5)
Hind-r2 primer 5'-CC introduced with camR-Nt primer and Hind III site using pLC87 as a templateAAGCTTAmplification was performed using GGCTTTTTGCAGGGCCAAGTCTTGTGTGTACCCT-3 ′. The amplified fragment was digested with Hind III and then subjected to polyacrylamide gel electrophoresis to separate and recover a 513 bp fragment. (This is the A fragment).
[0024]
After cleaving pLC87 with Eco RI, agarose gel electrophoresis was performed, and the 1383 bp fragment obtained by separation and recovery was converted into pBluescript II SK.^-Inserted into the Eco RI site of (STRATAGENE). The obtained plasmid pSK + LC87 was digested with Eco RI and Sph I and then subjected to polyacrylamide gel electrophoresis to separate and recover a 626 bp fragment. The fragment recovered from the gel was used as the first template for the introduction of a Hind III site and 4 amino acid (glycine) residues 5 ′ by the following 5 successive PCRs. First PCR is Hin 21 primer 5'-GGTGGAGGTGGAATGGATen cycles were amplified using CATCAAGCAGAGCTTGCTGCACGCA-3 ′ and RV primer 5′-CAGGAAACAGCTATGAC-3 ′. Second PCR is Hin 22 primer 5'-ATCGAGGAGGAGGGTGGAGGTGGAATGGACATCAAGCAGAGC-3 ′ and RV primer were used for 10 cycles using 1 μl of the first PCR reaction solution as a template. 3rd PCR is Hin 23 primer 5'-GCGCTCTTCAATATCGAGGAGGAGGGTGGAGGTGGAUsing ATGGAC-3 ′ and RV primer, 10 cycles were performed using 1 μl of the second PCR reaction solution as a template. 4th PCR is Hin 24 primer 5'-ATTACCTTGGTCGCGCTCTTCAATATCGAGGAGGAGGGTGGAUsing -3 ′ and RV primers, 10 cycles were performed using 1 μl of the third PCR reaction solution as a template. 5th PCR is Hin 25 primer 5'-CCAAGCTTUsing GACATTACCTTGGTCGCGCTCTTCAATATCGAG-3 ′ and RV primer, 25 cycles were performed using 1 μl of the 4th PCR reaction solution as a template. The amplified fragment was cleaved with Hind III and Bgl II, and subjected to polyacrylamide gel electrophoresis to separate and recover a 183 bp fragment. (This was called B-1 fragment)
[0025]
pSK + LC87 was cleaved with Bgl II and Eco RI, and subjected to polyacrylamide gel electrophoresis to separate and recover a 905 bp fragment. (This was called B-2 fragment)
[0026]
The B-1 and B-2 fragments were ligated and inserted into the Eco RI and Hind III sites of pBluscript II KS-. The obtained plasmid pKS + Hin25 was cleaved with Eco RI and Hind III and subjected to agarose gel electrophoresis to separate and recover a 1088 bp fragment. (This was C fragment)
[0027]
pMal-C2 was cut with HindIII and then blunted using Klenow DNA polymerase. This was self-ligated and the plasmid that disrupted the Hind III site was introduced into E. coli. A plasmid was prepared from this transformant to obtain pMal-C2 / Hind IIIb.
[0028]
The A fragment and the C fragment were ligated and inserted into the Xmn I and Eco RI sites of pMal-C2 / Hind IIIb. The obtained plasmid was used as p2xcamR as a plasmid for mass expression of 2xCamR.
[0029]
3) Construction of 3xcamR (Figs. 6-7)
PCR was performed using pKS + Hin25 as a template and using Hinr2 primer and RV primer. The amplified fragment was cleaved with Hind III, and subjected to polyacrylamide gel electrophoresis to separate and recover a 565 bp fragment. The recovered fragment was inserted into the Hind III site of pBluscript II KS-. PKS + Hin25-Hinr2 was cleaved with Hind III, and the resulting plasmid was subjected to polyacrylamide gel electrophoresis to separate and recover a 565 bp fragment. (This was D fragment)
[0030]
The D fragment was inserted into the Hind III site of p2xcamR. The resulting plasmid in the forward direction was used as a plasmid for mass expression of 3xCamR as p3xcamR.
[0031]
4) PCR
PCR was performed under the following conditions unless otherwise noted. The PCR reaction was performed at 94 ° C for 2 minutes for 1 cycle, 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 70 ° C for 1 minute for 25 cycles. Each (adenosine, thymine, guanosine, cytosine) deoxynucleotide triphosphate was added to a final concentration of 200 μM. 20 pmol of each primer was used. MgCl₂Was added to a final concentration of 1 mM. As a DNA polymerase, 1 unit of KOD DNA polymerase (TOYOBO CO., LTD) was used. 1 ng of plasmid DNA for each template was used.
[0032]
(2)CamR From Escherichia coli, which has a plasmid for mass production of CamR Purification
1) Purification of maltose binding protein (MalE) and CamR protein fusion protein
Each plasmid for mass production of CamR was introduced into E. coli XL1-blue. The obtained recombinants were named XL1-blue + 1xCamR, XL1-blue + 2xCamR, and XL1-blue + 3xCamR, respectively, and used for the following experiments.
[0033]
Each transformant stored in a glycerol solution at -85 ° C was streaked on a 1xLB agar medium containing 100 µg / ml ampicillin (Amp) and incubated at 37 ° C overnight. Single colonies were inoculated into 1 ml LB liquid medium containing 20 ml of Amp and cultured overnight at 37 ° C with shaking. The next day, 10 ml of the culture solution was transferred to 1 L glucose-rich 1 × LB medium containing Amp and cultured at 30 ° C. with shaking at 110 rpm until the OD600 nm reached 0.5. IPTG was added so that the final concentration was 0.3 mM, and further cultured for 2 hours. The culture was transferred to a 500 ml tube and collected by centrifugation at 4 ° C, 5,000 rpm for 5 minutes. The medium was discarded, and the cells were transferred to a 50 ml tube and stored at -85 ° C until the next operation. The cells were suspended in 1 ml column buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol) using a Pasteur pipette. Thereafter, the cells were transferred to a 10 ml volume plastic crusher tube. The mixture was sonicated for 20 seconds and cooled for 1 minute. This operation was performed 10 times. The cell disruption solution was transferred to a 1.5 ml microtube and centrifuged at 4 ° C., 15,000 rpm for 20 minutes. The supernatant (crude extract) was transferred to a new microtube. 15 ml of amylose resin (New England biolabs, Inc) was poured into a 2.5 x 10 cm column. The column was washed with 400 ml column buffer. The crude extract (2.5 μg / μl) was loaded at a flow rate of about 1 ml / min. Washed with 600 ml column buffer. The fusion protein was eluted with 60 ml of column buffer containing 10 mM maltose. Fractions were collected in 3 ml portions. Fractions containing protein were pooled. The protein was quantified using protein assay kit I (BIO-RAD Laboratories), and an appropriate amount was used for the factor Xa digestion.
[0034]
2) Purification of CamR protein from MalE and CamR fusion protein
Purification by ion exchange column chromatography
1 ml of a 1 mg / ml protein solution was transferred to a 1.5 ml microtube, 4 μg Factor Xa was added, and the mixture was incubated overnight at 16 ° C. for digestion. The digested 10 mg protein was replaced with TNbuffer (20 mM Tris-HCl, 25 mM NaCl, pH 8.0) using Ultracent-10. 6 ml of TSKgel DEAE-Toyopearl 650 Medium (Tosoh Corp.) was added to a 50 ml tube. To this, 20 ml of TNbuffer was added and mixed. The mixture was allowed to stand at room temperature for 1 hour and separated. The supernatant was discarded and repeated. The 1x10 cm column gave a 5 ml bed volume and was poured with DEAE resin. The column was washed with 15 ml TNbuffer. Protein digested with factor Xa and exchanged with buffer was added to the column. The column was washed with 30 ml of the same buffer. The column was gradient from an initial buffer concentration of 25 mM NaCl to a final buffer concentration of 500 mM NaCl (25 ml each containing 20 mM Tris-HCl, pH 8.0). Fractions were collected 1 ml each. The recovered protein was exchanged to a column buffer using Ultracent-10 and concentrated. Protein was quantified using Protein Assay Kit I (BIO-RAD Laboratories), then dispensed into 0.5 ml microtubes and stored at -85 ° C until used for SPR experiments.
[0035]
(3)SPR Method for measuring camphor
1) SPR device
Devices that measure the interaction of biomolecules using the surface plasmon resonance method are manufactured and sold by several machine manufacturers. In this example, NL-SPR670-E (1-channel batch type) manufactured by Nippon Laser Electronics Co., Ltd. was used. Hereinafter abbreviated as SPR device. This SPR device uses semiconductor laser light having high sensitivity as a light source. In addition, it is possible to select an SA membrane method using a disulfide compound, a thiol membrane method, an LB membrane method, or the like for immobilization of a ligand. When the SA membrane method using a disulfide compound is used to immobilize the ligand, the immobilization is easy, and the gold-deposited glass substrate, which is the sensor chip, can be reused, so there is no running cost. Gold-deposited glass substrates are available from the manufacturer. Therefore, in this example, the SA membrane method using a disulfide compound was adopted for immobilization of the ligand. The present embodiment can be applied to other SPR devices as long as DNA can be immobilized.
[0036]
2) Preparation of SA membrane using disulfide compound
The SA film using a disulfide compound was produced as follows according to the method of Nippon Laser Electronics Co., Ltd.
[0037]
(i) Acetone was added to a glass plate having a diameter of 10 ml. A gold-deposited glass substrate, which is a sensor chip, was immersed in this, and gently stirred and washed. (ii) A 10 mM 4,4-Ditho butyric acid (DBA) ethanol solution was further diluted 1000 times with ethanol to a final concentration of 10 μM. The diluted solution was added to another glass plate. A sensor chip washed with acetone was immersed in this and gently stirred. Then, it left still at room temperature for 30 minutes. By this reaction, the disulfide compound is bonded to the gold surface. (iii) Ethanol was added to another glass plate. The sensor chip of (ii) was immersed in this, gently stirred and washed. Ethanol was discarded and repeated. (iv) A solution of 25 mg aqueous carbodiimide and 15 mg N-hydroxysuccinimide dissolved in 10 ml 90% 1,4-dioxane was prepared and added to another glass plate. The sensor chip (iii) washed with ethanol was immersed in this, and gently stirred. Then, it left still at room temperature for 15 minutes. By this reaction, the sensor chip is activated so that a ligand having an amino group can be bound thereto. (v) Add ultrapure water to another glass plate. This was immersed in the sensor chip activated as described above, gently stirred and washed. (vi) The sensor chip washed with ultrapure water was placed on a paper towel and dried until it was attached to the SPR device.
[0038]
3) Preparation of operator DNA to be immobilized on the sensor chip
(i) Operator DNA sequence
The operator DNA sequence site to which the CamR repressor protein binds has been identified by Aramaki H., et al., (Supra). In this example, 27 bases out of this site were used as operator sequences. The sense strand is 5'-GCTCAGTATATCGCAGATATAGGGCCT-3 'and the antisense strand is 5'-AGGCCCTATATCTGCGATATACTGAGC-3'. These two oligonucleotide DNAs were introduced with an amino group at the 5 ′ end during synthesis so that they could be chemically bound to the activated sensor chip. The synthesized oligonucleotide DNA was purified by HPLC. Oligonucleotide DNA was synthesized under the same conditions as that produced by Japan Genosys Biotechnology Co., Ltd.
[0039]
(ii) DNA annealing
5 μl each of 50 pmol of the sense strand and antisense strand oligonucleotide DNA was dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). This was added to a 0.5 ml microtube, mixed, and heated at 95 ° C. for 10 minutes for denaturation. Then, it was allowed to stand at room temperature for 20 minutes for annealing. The annealed DNA was bound to the SA membrane by the method described later.
[0040]
4) Binding of purified CamR protein and DNA immobilized on sensor chip
In a 0.5 ml microtube, each 9 μl of purified CamR-MalE fusion protein (1xCamR-MalE, 2 x CamR-MalE, 3xCamR-MalE) or 1 μl of 10 x 10 x CamR binding buffer (200 mM HEPES (pH 8.0), 10 mM MEDTA, 100 mM 2-mercaptoethanol, 50 mM CaCl₂) And 2 μl of column buffer. This solution was allowed to stand for 10 minutes or more at room temperature until it was put on the SPR apparatus. The solution was added to a sensor chip on which DNA was immobilized.
[0041]
5) Binding reaction between purified CamR protein and camphor
A camphor ethanol solution prepared by dissolving in 100 mg / ml in ethanol was serially diluted to 100 fg / ml with a column buffer in which CamR protein was dissolved. 9 μl of purified CamR protein (2 × CamR) was added to a 0.5 ml microtube. Next, add each concentration of camphor (from 1 μg / ml to 100 fg / ml) diluted with 1 μl of 10 x CamR binding buffer and 2 μl of binding buffer, mix and mix at room temperature for at least 10 minutes. I left it until I put it. As a control, a sample to which only equal amounts of CamR protein (2 × CamR) and column buffer were added was used. This was added to a sensor chip on which DNA was immobilized.
[0042]
6) Operation procedure of SPR device
(i) The SPR device was turned on and stabilized one hour before use. (ii) Activated sensor chip was placed in the center of the prism of the device with a drop of refraction liquid for microscope (Cargille, refractive index 1.518) and placed gently, and fixed so that it does not move with a clasp. . (iii) 20 μl of TE was added. It was allowed to stand for about 1 minute until the resonance angle was stabilized. (iv) 10 μl of TE was sucked and discarded with a micropipette, and 10 μl (100 pmol) of the annealed DNA solution was added. The resonance angle increased, and then the mixture was allowed to stand for about 3 minutes until a certain resonance angle was reached. (v) 10 μl of the DNA solution was sucked with a micropipette and discarded, and 20 μl of column buffer was added. The resonance angle increased, and then the mixture was allowed to stand for about 3 minutes until a certain resonance angle was reached. (vi) 20 μl of column buffer was sucked with a micropipette and discarded, and 5 μl of 10 mg / ml BSA (bovine serum albumin dissolved in ultrapure water) solution dissolved in the column buffer was added. The resonance angle increased, and then the mixture was allowed to stand for about 5 minutes until a certain resonance angle was reached. Here, BSA was added to block non-specific binding of CamR protein by blocking empty sites on the chip surface where DNA was not bound. (vii) BSA solution was sucked with a micropipette and discarded. Add 10 μl of glycine-HCl (pH 1.0) solution, let stand for about 30 seconds, quickly aspirate 10 μl with a micropipette and discard, and pipette 10 μl at a time until the resonance angle becomes stable with the column buffer. Went. Glycine-HCl (pH 1.0) solution was added to remove free BSA. The resonance angle decreased to a certain line and was allowed to stand until stable. (viii) Next, 10 μl was sucked and discarded with a micropipette, and 10 μl of 1 × CamR binding buffer was added. The resonance angle increased, and then the mixture was allowed to stand for about 1 minute until a certain resonance angle was reached. The resonance angle at this time was recorded as a value before adding the CamR protein. (ix) 10 μl of the CamR protein sample prepared in 4) or 5) was added. The resonance angle increased, and then left to stand for about 1 minute until a certain resonance angle was reached. The resonance angle at this time was recorded as a value after adding CamR protein. (x) 10 μl was sucked with a micropipette and discarded, 10 μl of glycine-HCl (pH 1.0) solution was added and allowed to stand for about 30 seconds. Immediately suck and discard 10 μl with a micropipette, and pipette 10 μl at a time until the resonance angle is stable, and exchange the buffer. The solution was then aspirated with a micropipette and discarded. A glycine-HCl (pH 1.0) solution was added to dissociate the CamR protein bound to DNA and regenerate the sensor chip.
[0043]
(xi) Using a sensor chip regenerated with a glycine-HCl (pH 1.0) solution, (viii), (ix), and (x) were repeated with another sample. (xii) Quantitative determination of camphor was performed by combining the SPR reaction data for a certain time from the addition of each concentration of camphor and CamR protein binding reaction sample to stabilization.
[0044]
7) Reuse of sensor chip
The sensor chip used for SPR was reused after washing and removing DNA, protein, camphor, etc. bound to the surface by ultrasonic cleaning according to the following procedure. (i) 100 ml of acetone was added to a 200 ml beaker, the sensor chip used for this was added, and a weight was placed on the beaker in the bath of an ultrasonic washing machine and washed for 20 minutes. (ii) After washing, the sensor chip was placed on a paper towel, dried and used for the next SPR experiment.
[0045]
(B) Result
The base sequences including the gene region encoding the maltose binding protein and CamR protein of the constructed plasmid are shown in FIGS.
[0046]
The CamR protein could be purified to a single molecule by the purification method described above.
[0047]
Fig. 15 shows that the annealed DNA was immobilized on a sensor chip using 100 pmol, and purified using a 10 mM amylose resin column (1xCamR-MalE, 2 x CamR-MalE, 3xCamR-MalE). ) Or a fusion protein after digestion with factor Xa, followed by SPR experiment using 10 mM CamR protein (2 × CamR) separated and purified using DEAE ion exchange column chromatography. The experiment was repeated three times using the same sample. The result of the continuous experiment using one sensor chip is shown. The contents of the operations for the numbers indicated by the arrows in FIG. 15 are as shown in Table 1 below.
[0048]
[Table 1]

[0049]
Figure 16 shows typical results of binding experiments for DNA of 1xCamR-MalE, 2 x CamR-MalE, and 3xCamR-MalE fusion proteins from the results shown in Figure 15, respectively. Is superimposed on a single graph.
[0050]
FIG. 17 shows the results of the binding shown in FIG. 15 for 1 x CamR-MalE, 2 x CamR-MalE, and 3 x CamR-MalE fusion proteins. The results are shown averaged.
[0051]
Figure 18 shows 100 pmol of annealed DNA immobilized on a sensor chip, each digested with 2 x CamR-MalE fusion protein or fusion protein purified using a 5 mM amylose resin column with factor Xa, followed by DEAE ions Fig. 6 shows the change in resonance angle with time when an SPR experiment was performed using 5 mM 2 x CamR protein separated and purified using exchange column chromatography. The experiment was repeated three times using the same sample. This is a continuous experiment using a single sensor chip. The contents of the operations for the numbers indicated by the arrows in FIG. 18 are as shown in Table 2 below.
[0052]
[Table 2]

[0053]
Fig. 19 shows the result of binding of 2 x CamR-MalE fusion protein and 2 x CamR to the DNA of the results shown in Fig. 18, averaging the results of three resonance angles over a period of time. is there.
[0054]
FIG. 20 shows the results of averaging three resonance angles over a period of time in a binding experiment of 5 mM and 10 mM 2 × CamR-MalE fusion proteins with different concentrations to DNA.
[0055]
Fig. 21 shows the results of Fig. 18, in which the quantitative properties of camphor were examined using purified 2 x CamR protein and camphor at concentrations of 100 fg / ml, 1 ng / ml, and 10 μg / ml. A typical example of the results is extracted, and a response curve over a certain period of time is superimposed on a single graph.
[0056]
Figure 22 shows the results shown in Figure 18. Using the purified 2 x CamR protein, camphor quantification was investigated using camphors at concentrations of 100 fg / ml, 1 ng / ml, and 10 μg / ml. In the experiment, three results of the resonance angle for a certain time are averaged.
[0057]
The CamR protein used for the sensor is cultured in a recombinant (XL1-blue + 1xCamR, XL1-blue + 2xCamR, XL1-blue + 3xCamR) containing the constructed plasmid for mass expression, and expressed in large quantities as a maltose-binding protein fusion protein. The system could be easily purified. CamR protein can be obtained indefinitely by this purification method. The purified CamR protein can be stored for a long time at -20 ° C or -85 ° C, and is stable for more than 2 weeks at 4 ° C.
[0058]
As shown in Fig. 18, the measurement of camphor by SPR was able to obtain data in real time, and the time required for this could be completed in less than 20 minutes from DNA fixation to detection . The sensor chip could be regenerated using a glycine-HCl (pH 1.0) solution and could be used repeatedly more than 10 times with a single chip. The sensor chip could be reused by ultrasonic cleaning.
[0059]
When the same sample was repeated, almost the same resonance angle was obtained, and reproducibility was obtained (FIGS. 15 and 18). The resonance angle of the control with only CamR protein without addition of camphor was larger than that with addition of camphor (1 ng / ml and 10 μg / ml) (FIGS. 21 and 22). From this result, it was shown that CamR binding to camphor inhibited DNA binding depending on the concentration of camphor, as predicted by the principle of this example.
[0060]
From FIG. 19, it was known that the CamR protein DNA binding property examined using SPR was stronger when using only the CamR protein separated from the state of the fusion protein with the MalE protein. This is considered to be because in the state of the fusion protein of CamR protein with MalE protein, the MalE protein inhibits the conformation for binding to DNA. From this, it was expected that the CamR protein used for SPR was cut with factor Xa and then separated and purified using DEAE ion exchange column chromatography to improve the detection sensitivity of odorous substances.
[0061]
From Fig. 20, when comparing the resonance angle of 5 mM and 10 mM 2xCamR-MalE fusion protein, the resonance angle of 10 mM is higher than that of 5 mM, and the higher the concentration of CamR protein, the higher the resonance angle. It was observed. This is consistent with the fact that the higher the concentration of the substance bound in the reaction analyzing the interaction between molecules using SPR, the greater the response.
[0062]
From FIG. 21 and FIG. 22, the odorant camphor could quantitatively detect concentrations between 10 pg / ml and 100 ng. The detection limit is between 100 fg / ml and 10 pg / ml, but accurate values are not obtained. This level of sensitivity is 10 to 100 times higher than humans can feel in the nose, and is equivalent to or better than when using a gas chromatograph mass spectrometer.
[0063]
The material DNA and CamR protein used in this example are substances, and are not limited by the handling or experimental location. The sensitivity was 1 ng / ml in the case of the operon of the luminescence gene operon of the microorganism and the detection limit of 10 pg / ml in the case of the SPR method of this study. ml. Therefore, the detection sensitivity using the SPR method in this study is 100 times higher than that using the luminescent gene. The measurement time required 20 hours when using a luminescent gene, but the method using SPR in this study is less than 20 minutes, and the measurement time can be greatly shortened.
[0064]
Example 2
(A) (1)CamR Of plasmid for mass production
1) Construction of His-tagged CamR (Fig. 23)
Htnt-2 primer 5′-ATGCATCACCATCACCATCACATGGACATCAAG-3 ′ for use in PCR was phosphorylated by treatment with T4 polynucleotide kinase. The first PCR was performed for 10 cycles using pLC87 as a template and using Htnt-1 primer 5′-ATCACCATCACATGGACATCAAGCAGAGCTTGCTG-3 ′ and r7 primer 5′-GAGGAACACCAGATGTCGTT-3 ′. The second PCR was performed for 25 cycles using 1 μl of the first PCR reaction solution as a template and phosphorylated Htnt-2 primer and r7 primer 5′-GAGGAACACCAGATGTCGTT-3 ′. The amplified fragment was cleaved with Eco RI and then subjected to polyacrylamide gel electrophoresis, and a 1058 bp fragment was separated and recovered. This fragment was inserted into the Xmn I and Eco RI sites of pMal-c2. The obtained plasmid was used as pHis-tagged camR and a plasmid for mass expression of His-tagged CamR.
[0065]
(2)CamR From Escherichia coli, which has a plasmid for mass production of CamR Purification
1) Purification of maltose binding protein (MalE) and CamR protein fusion protein
The plasmid for mass production of CamR was introduced into E. coli XL1-blue. The obtained recombinant was named DH5α + His-tagged CamR, and then cut into MalE and CamR by Factor Xa in the same manner as in Example 1.
[0066]
2) Purification by Ni-NTA column chromatography
1 ml of a 1 mg / ml protein solution was transferred to a 1.5 ml microtube, 4 μg of Factor Xa (New England biolabs, Inc) was added, and the mixture was incubated overnight at 16 ° C. for digestion. Digested 10 mg of protein was prepared using Centriplus 10 (Amicon, Inc.) and lysis buffer (50 mM NaH₂PO_Four pH 8.0, 300 mM NaCl, 10 mM imidazole). 5 ml of Ni-NTA Superflow resin (QIAGEN) was poured onto a 1x10 cm column. The column was equilibrated with 40 ml lysis buffer. Protein exchanged into lysis buffer was added to the column at a flow rate of 0.5 ml / min. The column was washed with 40 ml lysis buffer. The column was washed with 40 ml wash buffer (50 mM NaH₂PO_Four, pH 8.0, 300 mM NaCl, 20 mM imidazole). 40 ml elution buffer (50 mM NaH₂PO_Four, pH 8.0, 300 mM NaCl, 250 mM imidazole) was added to elute His-tagged CamR. Fractions were collected 1 ml each. The recovered protein was exchanged with a column buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 10 mM β-mercaptoethanol) using Centriplus 10 (Amicon, Inc.) and concentrated. Protein was quantified using Protein Assay Kit I (BIO-RAD Laboratories), then dispensed into 0.5 ml microtubes and stored at -85 ° C until used for SPR experiments.
[0067]
(3)By camphor CamR Dissociation experiment
In the same manner as in Example 1, a series of operations of DNA fixation → buffer exchange → blocking with BSA → washing free BSA with glycine HCl (pH 1.0) → equilibrium with CamR binding buffer was performed. Then 10 μl of 10 mM CamR solution was added. When stabilized, 2 μl of camphor having a concentration of 1 pg / ml to 100 ng / ml, which was obtained by diluting 20 mM camphor solution dissolved in ethanol with column buffer, was added. As a control, a sample to which 2 μl of column buffer was added was used. This experiment was performed twice.
[0068]
(Four) CamR Monitoring experiment of dimer formation
In the same manner as in Example 1, a series of operations of DNA fixation → buffer exchange → blocking with BSA → washing free BSA with glycine HCl (pH 1.0) → equilibrium with CamR binding buffer was performed. Thereafter, 10 μl of 10 mM CamR solution was added and allowed to stand for 20 minutes or more.
[0069]
(Five) Musty odor substances 2-MIB Quantification of
As with camphor, experiments were conducted to determine the quantitativeness and detection limit of the musty odorous substance 2-methylisoborneol (2-MIB) in drinking water. 2-MIB was dissolved in ethanol to prepare a stock solution having a concentration of 20 mg / ml. This stock solution was diluted with distilled water to a concentration of 1 pg / ml to 10 μg / ml. In the experiment, 10 μl of a mixture of this 2-MIB dilution and 10 mM His-tagged CamR was used.
[0070]
(B) Experimental results
1) The nucleotide sequences including the gene region encoding the maltose binding protein and CamR protein of the constructed plasmid are shown in FIGS.
[0071]
2) Purification of CamR from the MalE-CamR fusion protein can be achieved easily by separating and purifying the protein expressed by adding 6 residues of histidine to the amino terminus of CamR using a Ni-NTA column. Could be separated and purified. Compared with the method of purification using an ion exchange column, this method has high purity and high recovery rate because there is no contamination with fusion protein left uncut by MalE or Factor Xa. In addition, the fusion protein with histidine added to the amino terminus was significantly digested by Factor Xa, and the remaining amount of the enzyme was reduced by a small amount of enzyme (1/5 of the previous amount). For this reason, the production and purification method of CamR according to the present method is advantageous in that the cost can be reduced because the amount of expensive Factor Xa used is small.
[0072]
3) In the experiment for examining the degree of dissociation of CamR from DNA by camphor, the value of decrease in resonance angle shown in FIG. 26 was used. As a result, when the camphor with a concentration of 1 pg / ml to 100 ng / ml was added, the decrease in the resonance angle was the same as or lower than the decrease in the control with buffer alone, and it did not increase significantly. For this reason, the dissociation phenomenon of CamR from DNA by addition of camphor could not be detected. Therefore, it was shown that the method of measuring the amount of odorous substances based on the phenomenon that CamR is dissociated from DNA by odorous substances cannot be applied (FIGS. 27 and 28). The contents of the operation for the numbers indicated by the arrows in FIG. 27 are as shown in Table 3 below.
[0073]
[Table 3]

[0074]
4) As for the binding property of CamR protein DNA, when a mixed sample in which CamR protein and camphor or 2-MIB were previously bound was bound to DNA, binding according to the amount of camphor or 2-MIB added was shown ( FIG. 31). In addition, the amount of change in the resonance angle in this case sufficiently captured the difference in the concentration of camphor or 2-MIB. Therefore, for detection of musty odor substances, a method of determining from the degree of inhibition of binding of CamR protein to DNA by musty odor substances is suitable. The basic principle of musty odor biosensor using SPR was completed by this method.
[0075]
5) His-tagged CamR has overwhelming DNA binding of the MalE-1xCamR fusion protein, His tagged CamR, MalE-2xCamR fusion protein, 2xCamR protein, and MalE-3xCamR fusion protein prepared in Example 1 or Example 2. Showed high DNA binding (FIG. 29). However, the control is a sample of only buffer without adding CamR, and the concentration is 2 mM of 2xCamR protein, and the other is 10 mM of protein. Moreover, a value is an average value of 3 times. This result is thought to be because the structure of CamR when linked to MalE or tandem inhibits DNA binding. For this reason, a single CamR protein purified by a Ni-NTA column was used in subsequent experiments.
[0076]
6) When the time-dependent change in the DNA binding state of the CamR protein was monitored by SPR, one upward-sloping DNA binding curve was detected until about 10 minutes, and then another upward-sloping curve was detected. (FIG. 30). Aramaki et al. (Supra) report that when CamR binds to DNA, it binds dimerically. Presuming from this consideration, the first reaction may be a reaction in which the first CamR binds to DNA, and the next reaction may be a reaction in which the second CamR binds to the first CamR and DNA. Absent. That is, the binding of CamR DNA does not bind to DNA after CamR becomes a dimer, but after one CamR binds, another CamR binds on the DNA. The first reaction is used to measure odorous substances in this study. However, if the odorous substance is measured using the second reaction in the future, the total reaction will increase, which may help improve sensitivity.
[0077]
7) When the musty odor substance 2-methylisoborneol (2-MIB) in clean water was measured by this sensor, it could be quantitatively detected from 1 ng / L to 10 mg / L (FIG. 31). This level is about several tens of times that of the human nose, which is similar to the gas chromatograph mass spectrometer (GC-MS) method.
[0078]
【The invention's effect】
As described above, according to the present invention, it is easy to handle compared to microorganisms, does not require culture, has a short measurement time, and measures substances such as odorous substances with higher sensitivity than conventional microorganism sensors. A sensor for measuring substances was provided.
[0079]
[Sequence Listing]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[Brief description of the drawings]
FIG. 1 is a schematic diagram for explaining the principle of a measurement method using a sensor of the present invention.
FIG. 2 is a diagram for explaining a method for constructing p1xcamR, which is a vector expressing a fusion protein of a camR protein monomer and MalE protein, which was prepared in an example of the present invention.
FIG. 3 is a diagram for explaining a method for constructing p2xcamR, which is a vector that expresses a fusion protein of a dimer of camR protein and a MalE protein produced in an example of the present invention.
FIG. 4 is a diagram showing a continuation of FIG. 3;
FIG. 5 is a diagram showing a continuation of FIG. 4;
FIG. 6 is a diagram for explaining a method of constructing p3xcamR, which is a vector expressing a fusion protein of a camR protein trimer and a MalE protein, which was prepared in an example of the present invention.
FIG. 7 is a diagram showing a continuation of FIG. 6;
FIG. 8 is a view showing a region encoding a fusion protein of a monomer of camR protein and a MalE protein in the p1xcamR vector and a base sequence in the vicinity thereof, together with an amino acid sequence encoded by the region.
FIG. 9 is a diagram showing a continuation of FIG. 8;
FIG. 10 is a view showing a region encoding a fusion protein of a dimer of camR protein and MalE protein in the p2xcamR vector and a base sequence in the vicinity thereof, together with an amino acid sequence encoded by the region.
FIG. 11 is a diagram showing a continuation of FIG. 10;
FIG. 12 is a view showing a region encoding a fusion protein of a camR protein trimer and a MalE protein in the p3xcamR vector and a base sequence in the vicinity thereof together with an amino acid sequence encoded by the region.
FIG. 13 is a diagram showing a continuation of FIG. 12;
FIG. 14 is a diagram showing a continuation of FIG. 13;
15 is a graph showing the relationship between time and resonance angle in an SPR experiment performed in Example 1. FIG.
FIG. 16 shows a typical example of the results of binding experiments to DNA of the same concentration of 1 × CamR-MalE, 2 × CamR-MalE, and 3xCamR-MalE fusion protein, respectively, with the same concentration. It is the figure which showed the response curve of time superimposed on one graph.
FIG. 17 is a graph showing the results of FIG. 15, in which binding of 1 × CamR-MalE, 2 × CamR-MalE, and 3 × CamR-MalE fusion protein to DNA was repeated three times at a certain resonance angle. It is the figure which averaged and showed the result. In the control, only the buffer used for protein dissolution was added.
FIG. 18 is a graph showing the relationship between time and resonance angle in an SPR experiment in which various concentrations of camphor were measured in an example of the present invention.
FIG. 19 is a graph showing an average of three results at a certain resonance angle in a binding experiment for 2 x CamR-MalE fusion protein and 2 x CamR DNA among the results shown in FIG. It is.
FIG. 20 shows averaged results of three resonance angles over a period of time in binding experiments of 5 mM and 10 mM 2 × CamR-MalE fusion proteins with different concentrations in DNA in an example of the present invention. FIG.
21. Among the results shown in FIG. 18, the quantitative properties of camphor were examined using camphor at concentrations of 100 fg / ml, 1 ng / ml, and 10 μg / ml using purified 2 × CamR protein. It is the figure which extracted the typical example of the result of the experiment, and overlapped and showed the response curve of a fixed time on one graph.
FIG. 22 shows the quantitative results of camphor using the purified 2 x CamR protein and the camphor concentrations of 100 fg / ml, 1 ng / ml, and 10 μg / ml. It is the figure which averaged and showed the result of 3 times of the resonance angle of a certain time in the experiment.
FIG. 23 constructs pHis-tagged CamR, which is a vector that expresses a fusion protein of a monomer of camR protein containing a tag consisting of 6 consecutive histidines and a MalE protein, produced in an example of the present invention. It is a figure for demonstrating a method.
FIG. 24 is a view showing a region encoding a fusion protein of a monomer of camR protein and a MalE protein in the pHis-tagged CamR vector and a base sequence in the vicinity thereof together with the amino acid sequence encoded by the region.
FIG. 25 is a diagram showing a continuation of FIG. 24;
FIG. 26 is a diagram showing a change in resonance angle before and after adding camphor to a sample solution in Example 2.
27 is a graph showing the relationship between time and resonance angle in the SPR experiment performed in Example 2. FIG.
28 is a graph showing the relationship between added camphor concentration and resonance angle in the SPR experiment performed in Example 2. FIG.
FIG. 29 is a view showing the binding properties between various repressor proteins and cam operon DNA prepared in Examples of the present invention.
30 is a graph showing the relationship between time and resonance angle in a CamR dimer formation monitoring experiment conducted in Example 2. FIG.
FIG. 31 is a graph showing the relationship between the concentration of 2-methylisoborneol and the resonance angle obtained by a 2-methylisoborneol measurement experiment performed in Example 2.

Claims (8)

A sensor for measuring a substance, wherein a nucleic acid that binds to a repressor protein that binds to a substance to be measured is bound to the surface of the sensor chip of the surface plasmon resonance device opposite to the side on which light is incident upon measurement. The nucleic acid has an operator sequence that binds to the repressor protein or a sequence in which one or more nucleotides are deleted, substituted, inserted, or added in the sequence and binds to the repressor protein. The sensor according to claim 1, which is a nucleic acid. The nucleic acid is a nucleic acid that binds to a camphor repressor protein, wherein the operator sequence of the camphor hydroxylase operon or a sequence in which one or more nucleotides are deleted, substituted, inserted or added in the sequence. 2. The sensor according to 2. The nucleic acid is a nucleic acid having a sequence in which the nucleotide sequence shown in SEQ ID NO: 1 or a plurality of nucleotides is deleted, substituted, inserted or added and binds to a camphor repressor protein. The sensor according to claim 3. The sensor according to claim 4, wherein the nucleic acid is a nucleic acid having a base sequence represented by SEQ ID NO: 1 in the sequence listing. The sensor according to any one of claims 1 to 5, wherein the substance to be measured is an odor substance. The sensor according to claim 6, wherein the odor substance is camphor, 2-methylisoborneol, isoborneol or geosmin. The sensor according to any one of claims 1 to 7, wherein the repressor protein is obtained by adding a tag comprising a plurality of histidine repeats to the amino terminus of a natural repressor protein.

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