JP3896460B2 - Non-natural protein production method, immobilization method and kit - Google Patents

Description

但し、Ｒ１はアジド基、アセチル基、アミノ基又は水酸基。
【００１９】
【化２】

但し、Ｒ２はアジド基、アセチル基又はアミノ基。
【００２０】
即ち、化１に示されるＹアナログは、チロシンの側鎖を構成するフェノール性芳香族環の３（メタ）位に置換基Ｒ１としてアジド基（−Ｎ₃）、アセチル基（−ＣＯＣＨ₃）、アミノ基（−ＮＨ₂）又は水酸基（−ＯＨ）を有している。化２に示されるＹアナログは、チロシンの側鎖を構成するフェノール性芳香族環の２（オルト）位に置換基Ｒ２としてアジド基、アセチル基又はアミノ基を有している。なお、これらのＹアナログの置換基は、メタ位又はオルト位のいずれに結合されていても構わないが、側鎖の端部側に位置しＹアナログ修飾体にする反応が進行しやすいことからメタ位に結合されているのが好ましい。また、これらのＹアナログは、Ｌ型又はＤ型のいずれの光学異性体であっても構わないが、蛋白質合成効率が高いことからＬ型であるのが好ましい。
【００２１】
Ｙアナログ修飾体は、前記Ｙアナログの側鎖（フェノール性芳香族環の２位又は３位）に修飾物が結合した有機化合物である。前記修飾物は、修飾基と、その修飾基に結合した反応基とを備えている。前記反応基としては、オルトアルコキシカルボニルアリールジアリールホスフィン（トリアリールホスフィン（triarylphosphine）誘導体）、ヒドラジン（hydrazine）、コハク酸イミドエステル（succimidyl ester）、イソチオシアン酸（isothiocyanate）塩、塩化スルホニル（sulfonyl chloride）、アルデヒド（aldehyde）、又はカルボン酸や酸ハライド若しくはその酸無水物が挙げられる。前記修飾基は、特に限定されないが、実用的な用途拡大が容易であることから、蛍光物質、疎水性の巨大分子、親水性物質、電荷を有する物質、水素結合能を有する物質、化学反応性を有する物質、生理活性物質（酵素等）、生体構成物質（蛋白質、糖鎖、ポリヌクレオチド、脂肪酸等）、ビオチン（Biotin）、抗体又はその一部等が好適に用いられる。また、修飾基として、ポリエチレングリコール等のドラッグデリバリーシステム（ＤＤＳ）に用いられる機能性担体を用いてもよい。
【００２２】
このＹアナログ修飾体は、Ｙアナログの置換基と、前記修飾物の反応基とを化学的又は生物化学的に結合（共有結合）させることにより得られる。
前記置換基と反応基との組合わせとしては、置換基がアジド基の場合には反応基としてはトリアリールホスフィン誘導体となり、図１に模式的に示されるような反応（Staudinger ligation反応）が行われる。また、置換基がアセチル基の場合には反応基がヒドラジンとなり、置換基がアミノ基の場合には反応基がコハク酸イミドエステル、イソチオシアン酸塩、塩化スルホニル、アルデヒド、又はカルボン酸や酸ハライド若しくはその酸無水物となる。これら置換基と反応基との反応は化学的反応である。また、前記Ｙアナログがドーパの場合には、そのフェノール性芳香族環の３位及び４位の水酸基をドーパオキシダーゼ（DOPA oxidase）による酵素反応を利用しつつ反応基としてのアミノ基と結合（生物化学的反応）させることにより、修飾物が結合してＹアナログ修飾体となる。これらのＹアナログの置換基を修飾する反応は、生理的条件下（例えば大腸菌や酵母等の一般細菌又は単細胞生物が生存可能な条件下）で行うことが可能となっている。
【００２３】
実施形態の第１の非天然蛋白質の製造方法は、Ｙアナログと、サプレッサーｔＲＮＡと、アミノアシルｔＲＮＡ合成酵素変異体とを含有する蛋白質合成系に、鋳型を加えて蛋白質合成させる蛋白質合成工程を行うものである。前記サプレッサーｔＲＮＡは、ｍＲＮＡ上の終止コドン（アンバー、オーカー又はオパール）をあるアミノ酸に対応するコドンとして読むナンセンスサプレッサーｔＲＮＡ又はミスセンスサプレッサーｔＲＮＡである。
【００２４】
前記アミノアシルｔＲＮＡ合成酵素変異体は、前記Ｙアナログを前記サプレッサーｔＲＮＡに特異的に結合させる酵素である。このアミノアシルｔＲＮＡ合成酵素変異体としては、前記Ｙアナログを基質とするものであれば特に限定されないが、標準型アミノ酸に対する基質特異性が低くなりやすく有用であることから野生型のチロシルｔＲＮＡ合成酵素を改変したチロシルｔＲＮＡ合成酵素変異体（TyrＲＳ変異体）が最も好適に用いられる。前記野生型のチロシルｔＲＮＡ合成酵素としては、配列番号１で表されるアミノ酸配列からなる酵母（Saccharomyces cerevisiae）チロシルｔＲＮＡ合成酵素が挙げられる。
【００２５】
この酵母チロシルｔＲＮＡ合成酵素の変異体、即ち酵母TyrＲＳ変異体としては、配列番号１で表されるアミノ酸配列の４３番目のＴｙｒがＧｌｙに置換されたＹ４３ＧチロシルｔＲＮＡ合成酵素変異体（Y43G TyrＲＳ）、配列番号１で表されるアミノ酸配列の８１番目のＨｉｓがＶａｌに置換されたＨ８１ＶチロシルｔＲＮＡ合成酵素変異体（H81V TyrＲＳ）、配列番号１で表されるアミノ酸配列の１８６番目のＧｌｎがＶａｌに置換されたＱ１８６ＶチロシルｔＲＮＡ合成酵素変異体（Q186V TyrＲＳ）、配列番号１で表されるアミノ酸配列の１８６番目のＧｌｎがＴｈｒに置換されたＱ１８６ＴチロシルｔＲＮＡ合成酵素変異体（Q186T TyrＲＳ）、又は配列番号１で表されるアミノ酸配列の１８６番目のＧｌｎがＩｌｅに置換されたＱ１８６ＩチロシルｔＲＮＡ合成酵素変異体（Q186I TyrＲＳ）が挙げられる。また、前記Y43G TyrＲＳ、H81V TyrＲＳ、Q186V TyrＲＳ、Q186T TyrＲＳ若しくはQ186I TyrＲＳの１〜３６４番目のアミノ酸配列からなる変異体も同様の酵素活性があることから用いられる。
【００２６】
さらに、このTyrＲＳ変異体としては、精製が容易であること等から、前記各TyrＲＳ変異体のＣ末端にヒスチジンタグ等のタグ（tag）が結合したものを用いるとよい。なお、前記Y43G TyrＲＳについては、S.Ohno et.al., Changing the Amino Acid Specificity of Yeast Tyrosyl-tRNA Synthetase by Genetic Engineering, J.Biochem.130,417-423(2001)に開示されている。また、前記各酵母TyrＲＳ変異体はいずれも、酵母ナンセンスサプレッサーｔＲＮＡ^Tyr（ｔＲＮＡ^Tyr）（アンバー、オーカー又はオパール）を基質とする。
【００２７】
前記鋳型は、前記サプレッサーｔＲＮＡが認識する終止コドンを、部位特異的突然変異（site-directed mutagenesis）等により人為的に翻訳領域に配したポリヌクレオチドであり、ＤＮＡ又はｍＲＮＡの形態で用いられる。前記ＤＮＡとしては、転写に必須な配列を含む二本鎖ＤＮＡが挙げられ、種々のベクターやプラスミド等に導入して利用するのが最も簡便である。なお、前記翻訳領域の３’末端に位置する終止コドンは、前記サプレッサーｔＲＮＡが認識する終止コドンとは異なる塩基配列のコドンが配される。
【００２８】
前記蛋白質合成系は、前記鋳型がコードする蛋白質を合成させるためのシステムであり、インビトロ（in vitro）蛋白質合成系又はインビボ（in vivo）蛋白質合成系が挙げられる。なお、この蛋白質合成系に含有されるアミノアシルｔＲＮＡ合成酵素変異体及びその変異体の基質としてのサプレッサーｔＲＮＡは、同蛋白質合成系の翻訳過程に関与する成分とは異なる種の生物に由来するものであるのが好ましく、一方が真核細胞由来で他方が原核細胞由来であるのがより一層好ましい。このとき、第１の非天然蛋白質にＹアナログを適切かつ部位特異的に組込むことがより一層容易となる。
【００２９】
インビトロ蛋白質合成系としては、鋳型としてのｍＲＮＡからの翻訳を行わせるインビトロ翻訳系、又は鋳型としてのＤＮＡから転写及び翻訳を行わせるインビトロ転写翻訳系が挙げられる。これらインビトロ蛋白質合成系としては、転写、若しくは転写及び翻訳に必要な成分を人工的に混合することにより構築されたもの、細胞抽出物からなる無細胞蛋白質合成系、又は両者を混合することにより構築されたものが用いられる。前記無細胞蛋白質合成系は、大腸菌等の原核細胞、酵母等の真核細胞、プロトプラスト等の植物細胞、昆虫細胞、哺乳動物細胞等の細胞を主として脱核することにより得られ、転写及び翻訳に必要な各種成分が高含有されている。このインビトロ蛋白質合成系としては、広く有用性が認められていることから、大腸菌Ｓ３０抽出物又は小麦胚芽抽出物を含有するもの、ウサギ網状赤血球の溶血系、チャイニーズハムスター卵巣細胞（ＣＨＯ）、バキュロウィルスベクター蛋白質発現系、アフリカツメガエル卵母細胞等が好適に用いられる。また、非天然アミノ酸の導入に負に働く因子（例えば核酸分解酵素、蛋白質分解酵素、解離因子、酸化・還元酵素等）を破壊（欠損）、又は免疫沈降等により取り除いた大腸菌の細胞抽出液も好適に用いられる。
【００３０】
このインビトロ蛋白質合成系中に含まれるＹアナログは、同蛋白質合成系中の各標準型アミノ酸の含有量よりも多いのが好ましく、より好ましくは前記含有量の１〜１００倍量、さらに好ましくは５〜１０倍量であるとよい。Ｙアナログが前記含有量の１倍量未満の場合にはＹアナログを非天然蛋白質に高い効率で組込ませることができず、逆に１００倍量を越える場合には蛋白質合成系の秩序が乱されて破綻しやすくなる。また、インビトロ蛋白質合成系中に含まれるサプレッサーｔＲＮＡは、同蛋白質合成系中の各ｔＲＮＡの含有量と同程度であるのが好ましく、より好ましくは前記含有量の０．０１〜１０倍量、さらに好ましくは０．５〜２倍量であるとよい。サプレッサーｔＲＮＡが前記含有量の０．０１倍量未満の場合にはＹアナログを非天然蛋白質に高い効率で組込ませることができず、逆に１０倍量を越える場合には蛋白質合成系の秩序が乱されて破綻しやすくなる。また、インビトロ蛋白質合成系中に含まれるアミノアシルｔＲＮＡ合成酵素変異体は、同蛋白質合成系中の各アミノアシルｔＲＮＡ合成酵素（野生型）の含有量よりも多いのが好ましく、より好ましくは前記含有量の０．０５〜０．５ｍｇ／ｍｌ、さらに好ましくは０．１〜０．２ｍｇ／ｍｌであるとよい。アミノアシルｔＲＮＡ合成酵素変異体が前記含有量の０．０５ｍｇ／ｍｌ未満の場合にはＹアナログを非天然蛋白質に高い効率で組込ませることができず、逆に０．５ｍｇ／ｍｌを越える場合には蛋白質合成系の秩序が乱されて破綻しやすくなる。
【００３１】
インビボ蛋白質合成系としては、外来遺伝子としての前記鋳型及びアミノアシルｔＲＮＡ合成酵素変異体が発現可能となるように形質転換された細胞（形質転換細胞）、又は該細胞を含む生物個体が用いられる。前記形質転換細胞としては、脱核されていない生細胞が用いられ、大腸菌等の原核細胞、酵母等の真核細胞、植物細胞、昆虫細胞、哺乳動物細胞等が好適に用いられる。なお、前記アミノアシルｔＲＮＡ合成酵素変異体の基質としてのサプレッサーｔＲＮＡが前記形質転換細胞中に内在されていない場合には、該サプレッサーｔＲＮＡも外来遺伝子として同細胞中に形質転換される必要がある。このインビボ蛋白質合成系では、Ｙアナログを食餌として摂取させることにより、前記外来遺伝子が発現された形質転換細胞中の蛋白質合成系が非天然蛋白質を継続的かつ大量に生産する。
【００３２】
一方、この第１の非天然蛋白質の製造方法に用いられるＹアナログとしては、蛋白質合成系に通常含有されていない置換基を有していることから、アジドチロシン又はドーパが好適に用いられる。また、特異性の高い酵素反応を利用して第２の非天然蛋白質を製造することが可能であることから、ドーパが特に好適に用いられる。
【００３３】
実施形態の第２の非天然蛋白質の製造方法は、前記第１の非天然蛋白質の製造方法における蛋白質合成工程を行った後、さらに後修飾工程を行うものである。この後修飾工程は、前記Ｙアナログの側鎖のアジド基、アセチル基、アミノ基又は水酸基を修飾してＹアナログ修飾体にする工程であり、上記Ｙアナログ修飾体における化学的反応又は生物化学的反応が行われる。なお、この第２の非天然蛋白質の製造方法において、前記蛋白質合成工程と後修飾工程との間に、蛋白質合成に関与しなかった未反応のＹアナログを取り除く操作を行うのが好ましい。
【００３４】
実施形態の非天然蛋白質の固定化方法は、前記第１の非天然蛋白質を固定化担体に対して固定化するものである。前記固定化担体は、水又は有機溶媒に不溶な固体からなる支持体と、その支持体に結合した反応基とを備えている。前記反応基は、上記Ｙアナログ修飾体の置換基と反応するもの（上記反応基と同じもの）が用いられる。そして、この固定化方法では、上記Ｙアナログ修飾体における化学的反応又は生物化学的反応と同じ反応を行うことにより、固定化担体に第１の非天然蛋白質を固定化させる。この非天然蛋白質が固定化された固定化担体は、例えばアフィニティクロマトグラフィー、プロテインチップ、バイオリアクター等に利用することができる。また、コラーゲン、プロテオグリカン、フィブロネクチン、エラスチン、ヒアルロン酸等の細胞外マトリックスや、カドヘリンファミリー、免疫グロブリンスーパーファミリー、インテグリンファミリー、セレクチンファミリー、リンク蛋白質ファミリー、シアロムチンファミリー等の細胞接着分子を固定化させたインプラント材や再生医療用人工素材にも利用することができる。
【００３５】
上記第一実施形態によって発揮される効果について、以下に記載する。
・ 第一実施形態の第１の非天然蛋白質は、蛋白質構成アミノ酸としてＹアナログが部位特異的に組込まれたものである。第一実施形態の第２の非天然蛋白質は、蛋白質構成アミノ酸としてＹアナログ修飾体が部位特異的に組込まれたものでる。即ち、これら非天然蛋白質は、蛋白質構成アミノ酸として非天然アミノ酸が部位特異的に組込まれていることから、天然蛋白質とは異なる性質を人為的に付与されたものである。
【００３６】
このため、これらの非天然蛋白質は、天然蛋白質とは分子構造が明確に区別されていることから、その生体内での局在や挙動を調べるのが容易である。特に、第１の非天然蛋白質では、Ｙアナログのサイズが標準型アミノ酸とほとんど相違しないことから、生きた生物体に対する悪影響が極めて少ないうえ、該生物体に対して後修飾工程を行ってインサイチュ（in situ）による可視化を行うことが極めて容易である。また、電気泳動後に標識化（可視化）することも容易である。また、これらの非天然蛋白質は、非天然アミノ酸が部位特異的に組込まれていることから、天然蛋白質の生理活性に関与する蛋白質構成アミノ酸を特定したり、蛋白質の立体構造解析（structurome）に利用したり、情報処理解析技術の開発に利用することができる。また、アフィニティクロマトグラフィー担体、プロテインチップやその解析機器、バイオリアクター、ナノテクノロジー、電子機器、情報解析、精密計測機器等の研究又は工業的な応用が容易である。また、ゲノム情報を活用した画期的な医薬品素材やバイオマーカーの開発等の医療関連分野にも応用することが可能である。特に、第１の非天然蛋白質は蛋白質合成系による生物学的な製造方法により得られ、第２の非天然蛋白質は置換基と反応基との化学反応等が生理的な条件下で行われていることから、その生理活性の変化や立体構造の変化を極めて寡少に止めることが容易であるうえ、人為的に変化させることも容易である。
【００３７】
・ 第一実施形態の第１の非天然蛋白質の製造方法では、Ｙアナログが用いられている。これらのＹアナログのアジド基、アセチル基、アミノ基又は水酸基は、いずれも標準型アミノ酸に対する分子構造の差を明確化しているうえ、いずれも立体障害をほとんど生じない小さいサイズのものである。このため、これらのＹアナログはいずれも、リボソーム上でアミノアシルｔＲＮＡがアミノアシルｔＲＮＡ結合部位（Ａ部位）に結合する反応を行う際の立体障害とはならないサイズであることから、蛋白質合成工程が天然蛋白質の場合とほぼ同様に迅速に進行する。さらに、これらのＹアナログはいずれも、セントラルドグマに従った蛋白質合成系を用いた生理的な反応により非天然蛋白質に組込まれることから、該蛋白質の生理活性の低下が著しく効果的に抑えられている。また、トリプレットからなる遺伝コードを利用していることから、公知の天然蛋白質をコードするポリヌクレオチドの翻訳フレームを部位特異的突然変異等により大幅に改変する必要がなく、極めて手軽に製造することが可能である。
【００３８】
・ 第一実施形態の第２の非天然蛋白質の製造方法は、蛋白質合成工程を行って第１の非天然蛋白質を製造した後、該蛋白質と修飾物とを混合して生理的条件下で反応（化学反応や酵素反応等）させる後修飾工程を行うことにより、同蛋白質に修飾物を結合させるものである。この製造方法では、蛋白質合成工程及び後修飾工程ともに生理的条件下で行われることから、第２の非天然蛋白質の生理活性の低下を極めて効果的に抑えることができる。さらに、前記後修飾工程における置換基と反応基との反応は、著しく迅速かつ効率的に進行する反応である。一方、実施形態の非天然蛋白質の固定化方法は、前記後修飾工程とほぼ同様な反応を利用していることから、全く同様な効果が奏され得る。
【００３９】
（第二実施形態）
以下、この発明の第二実施形態を上記第一実施形態を参照しつつ説明する。
第二実施形態の非天然蛋白質は、Ｙアナログとしてのブロモチロシン（bromo tyrosine）が部位特異的に組込まれたポリペプチドである。この非天然蛋白質は、Ｘ線を好適に散乱させる性質を有するブロモ基（−Ｂｒ）が部位特異的に組込まれていることから、その非天然蛋白質のＸ線結晶構造解析に好適に利用される。さらに、この非天然蛋白質は、前記ブロモ基が極めて立体障害の少ない置換基であることから、該非天然蛋白質の立体構造（三次構造、四次構造）と天然蛋白質のそれとの間に相違が生じにくく、極めて好適な構造解析を行うことができる。
【００４０】
前記ブロモチロシンは、チロシンの側鎖を構成するフェノール性芳香族環の３（メタ）位又は２（オルト）位に置換基としてのブロモ基を備えたＹアナログであり、上記化１に示されるＹアナログの置換基Ｒ１をブロモ基としたもの又は上記化２に示されるＹアナログの置換基Ｒ２をブロモ基としたものが挙げられる。なお、このブロモチロシンのブロモ基は、メタ位又はオルト位のいずれに結合されていても構わない。また、このブロモチロシンとしては、Ｌ型又はＤ型のいずれの光学異性体であっても構わないが、蛋白質合成効率が高いことからＬ型であるのが好ましい。
【００４１】
第二実施形態の非天然蛋白質の製造方法は、上記第一実施形態の第１の非天然蛋白質の製造方法と全く同様に行われる。
第二実施形態のキットとしての非天然蛋白質の製造用キットは、少なくともブロモチロシン及び該ブロモチロシンをサプレッサーｔＲＮＡに結合させるアミノアシルｔＲＮＡ合成酵素変異体が含有されており、さらにサプレッサーｔＲＮＡが含有されているのがより好ましい。また、この製造用キット中には、さらに蛋白質合成系が含有されているのが好ましい。なおこのとき、前記サプレッサーｔＲＮＡ及びアミノアシルｔＲＮＡ合成酵素変異体は、該蛋白質合成系の翻訳過程に関与する成分とは異なる種の生物に由来するものであるのが好ましく、一方が真核細胞由来で他方が原核細胞由来であるのがより一層好ましい。また、この製造用キット中に、部位特異的突然変異を行うための遺伝子キットが含有されていてもよい。
【００４２】
上記第二実施形態によって発揮される効果について、以下に記載する。
・ 第二実施形態の非天然蛋白質は、蛋白質構成アミノ酸としてブロモチロシンが部位特異的に組込まれたものである。このため、この非天然蛋白質は、ブロモチロシンのサイズが標準型アミノ酸とほとんど相違しないことから、非天然蛋白質の三次構造が天然蛋白質のフォールディング様式とほとんど相違することはない。従って、Ｘ線結晶構造解析において立体構造を調べるために好適に用いられるうえ、その際にはＸ線の散乱特性に非常に優れていて好ましい。さらに、ブロモチロシンが部位特異的に組込まれていることから、前記従来のセレノメチオニン置換タンパク質の場合のような種々の不具合の発生がほとんどない。
【００４３】
・ 第二実施形態の非天然蛋白質の製造方法では、Ｙアナログとしてブロモチロシンが用いられている。このＹアナログのブロモ基は、立体障害をほとんど生じない小さいサイズのものであることから、上記第一実施形態の第１の非天然蛋白質の製造方法と全く同様の効果を奏する。
【００４４】
【実施例】
以下、前記実施形態を具体化した実施例及び比較例について説明する。
＜インビトロ蛋白質合成系（大腸菌Ｓ３０抽出物）の調製＞
ＬＢ（Luria-Bertani）プレートに生やした大腸菌Ｑ１３株（国立遺伝学研究所より分譲）の単一コロニーをＬＢ培地１００ｍｌに植菌し、３７℃で一晩振盪培養（プレ培養）した。次に、前記プレ培養後の培養液を１００倍量の２×ＹＴ（Trypton Pepton,Yeast Extract）培地（DIFCO社製）に加え、６００ｎｍにおける濁度（ＯＤ₆₀₀）が０．３〜０．４になるまで３７℃で振盪培養（本培養）した。続いて、前記本培養後の培養液を氷冷し、すぐに遠心分離して集菌した後、その菌体を、ヘペス緩衝液（Hepes-KOH(pH7.5) 20mM）、酢酸マグネシウム（10mM）、酢酸アンモニウム（20mM）、2-メルカプトエタノール（10mM）を含む溶液４０ｍｌに懸濁して菌体懸濁液を得た。前記菌体懸濁液にLysozyme（生化学工業社製）を２ｍｇ／ｍｌになるように加え、氷上に約１０分間静置した。続いて、前記菌体懸濁液を遠心分離して沈澱を得た後、その沈澱（菌体）の重量を量り、その重量と同量のＳ３０緩衝液（菌体１ｇに対し１ｍｌのＳ３０緩衝液）で懸濁してＳ３０懸濁液を得た。そのＳ３０懸濁液を細胞破砕した後、遠心分離することにより上清を得た。この上清は、インビトロ蛋白質合成系としての大腸菌Ｓ３０抽出物であり、小分けして液体窒素で長期間保存することが可能である。
【００４５】
＜非天然蛋白質の製造＞
ヘペス緩衝液（Hepes-KOH(pH7.6) 50mM）、酢酸マグネシウム（7.7mM）、酢酸アンモニウム（27.5mM）、ジチオスレイトール（DTT 1.7mM）、ヌクレオチド三リン酸（ATP 1.25mM,GTP 0.83mM,UTP 0.83mM,CTP 0.83mM）、クレアチンリン酸（80mM）、クレアチンキナーゼ（0.21mg/ml）、Ｔ７ＲＮＡポリメラーゼ（本発明者らが単離0.1mg/ml）、大腸菌Ｑ１３株ｔＲＮＡミックス（本発明者らが単離 3.4A_260unit/ml）、Ｌ−チロシン（80μM）、Ｌ−チロシン以外の標準型アミノ酸１９種（each 160μM）、ホリン酸（50μM）、酢酸カリウム（200mM）、ポリエチレングリコール（wt.8000 4%）、３−アジド−Ｌ−チロシン（本発明者らが合成 500μM）、酵母Y43G TyrＲＳ（本発明者らが作製 0.17mg/ml）、酵母サプレッサーｔＲＮＡ^Tyr(CUA)（本発明者らが単離 0.1A_260unit/ml）、鋳型ＤＮＡ（20μg/ml）、前記大腸菌Ｓ３０抽出物（30%(v/v)）及び脱イオン水を、括弧内に記載された終濃度となるように試験管内に入れて混合した後、３０℃で１時間インキュベートした。
【００４６】
比較例１は、鋳型ＤＮＡとして、野生型の大腸菌ジヒドロ葉酸還元酵素（dihydrofolate reductase［EC:1.5.1.3］；ＤＨＦＲ）遺伝子、即ち配列番号２で表される塩基配列からなる二本鎖ＤＮＡが組込まれたプラスミドベクターpET 21-a(+)（NOVAGEN社製）を使用した。比較例２は、鋳型ＤＮＡとして、前記プラスミドベクターpET 21-a(+)を使用した。実施例１は、鋳型ＤＮＡとして、大腸菌ＤＨＦＲの３７位のＡｓｎに対応するコドンを部位特異的突然変異によりアンバーコドンに改変した遺伝子、即ち配列番号２で表される塩基配列の１０９〜１１１番目をtagに改変した二本鎖ＤＮＡが組込まれたプラスミドベクターpET 21-a(+)を使用した。また、実施例２は、鋳型ＤＮＡとして、大腸菌ＤＨＦＲの１２８位のＴｙｒに対応するコドンを部位特異的突然変異によりアンバーコドンに改変した遺伝子、即ち配列番号２で表される塩基配列の３８２〜３８４番目をtagに改変した二本鎖ＤＮＡが組込まれたプラスミドベクターpET 21-a(+)を使用した。
【００４７】
次に、前記インキュベート後の反応液をＮｉ−ＮＴＡスピンカラム（Qiagen社製、溶出液はｐＨ７．４のリン酸緩衝液）により蛋白質合成に関与しなかった３−アジド−Ｌ−チロシンを除去しつつ精製した後、蛍光標識された修飾物を終濃度２５０μＭとなるように添加して３７℃で１時間インキュベートした。続いて、これら実施例１，２、比較例１，２及び予め単離された野生型の大腸菌ＤＨＦＲをＳＤＳゲル電気泳動した後、クマシーブリリアントブルー（ＣＢＢ）染色及び蛍光染色を行った。なお、前記修飾物は、本発明者らが合成したトリアリールホスフィン誘導体であり、図１に示されるような構造を有している。
【００４８】
その結果、比較例１，２では蛍光が検出されなかったが、改変した鋳型ＤＮＡを用いて製造された実施例１，２では、いずれも野生型の大腸菌ＤＨＦＲと同じ分子量のＣＢＢ染色バンドの位置に蛍光バンドが検出された。また、データは示さないが、実施例１，２の非天然蛋白質ではいずれも、野生型の大腸菌ＤＨＦＲとほぼ同程度のＤＨＦＲ活性が確認された。
【００４９】
＜非天然蛋白質の固定化＞
アミノコーティングプレート（住友ベークライト社製）の各穴（ウェル）に、トリアリルフォスフィン誘導体（5mg/ml ジメチルスルホキシド溶液）を分注し、３７℃で２時間インキュベートして吸着させた。各ウェルをリン酸緩衝液(pH7.4)で洗浄した後、前記実施例１，２、比較例１，２及びリン酸緩衝液（コントロール）を各ウェルに分注し、３７℃で６時間インキュベートした。次に、各ウェルをリン酸緩衝液(pH7.4)で洗浄した後、テキサスレッドで標識したメトトレキセートを各ウェルに分注し、３７℃で２時間インキュベートした。最後に、各ウェルをリン酸緩衝液(pH7.4)で洗浄した後、イメージングアナライザー（アマシャムファルマシア・バイオテク社製）で解析したところ、実施例１，２を分注したウェルでは底面及び内壁面にテキサスレッドの色が明確に確認された。一方、比較例１，２及びリン酸緩衝液を分注したウェルでは確認されなかった。
【００５０】
＜アミノアシルｔＲＮＡ合成酵素変異体の基質特異性の調査＞
Y43G TyrＲＳ、H81V TyrＲＳ、Q186V TyrＲＳ、Q186T TyrＲＳ及びQ186I TyrＲＳの５種類の酵母TyrＲＳ変異体について、各種Ｙアナログに対する基質特異性を酸性条件下ポリアクリルアミドゲル電気泳動により確認した。なお、前記電気泳動は、Nucleic Acids Symposium Series No.35, 285-286(1996) (Oxford University Press, ISSN 0261 3166)の方法に従って行われた。結果を表１に示す。なお、表１において、酵母TyrＲＳがＹアナログを認識したことを実際に確認した場合には◎と表記した。また、酵母TyrＲＳがＹアナログを認識したことを直接確認してはいないが、野生型の酵母チロシルｔＲＮＡ合成酵素（酵母TyrＲＳ）等の試験結果から、認識する可能性が極めて高い場合には○、認識する可能性が十分にある場合には△、認識する可能性が極めて低い又は認識しない場合には×、認識するか否か全く不明の場合にはＮＤと表記した。
【００５１】
【表１】

その結果、これら５種類の酵母TyrＲＳ変異体については、全ての確認が取れていない状況ではあるが、基質特異性に多少の相違は見られるが概ね上記各実施形態の非天然蛋白質の製造に使用できることが確認された。さらには、これら５種類の酵母TyrＲＳ変異体は、メタ位（３位）に置換基を有するものは表１の通りであるが、野生型の酵母TyrＲＳがオルト位（２位）に置換基を有するものを基質として認識するという事実から同様に認識する可能性が高い。一方、Y43G TyrＲＳ及びH81V TyrＲＳについては、Ｄ−チロシンを認識しないことからＤ型のＹアナログを認識する可能性は著しく低いが、Ｄ−ドーパは認識する可能性は高い。Q186V TyrＲＳ、Q186T TyrＲＳ及びQ186I TyrＲＳについては、Ｄ−チロシンを基質として認識する可能性が高いが、Ｄ−ドーパは基質とはなる可能性は極めて低い。
【００５２】
なお、上記実施形態は、次のように変更して具体化することも可能である。
・ 第一実施形態の第１の非天然蛋白質の製造方法において、まず、Ｙアナログと、サプレッサーｔＲＮＡと、アミノアシルｔＲＮＡ合成酵素変異体とを予め酵素反応させることにより、Ｙアナログがアミノアシル化されたサプレッサーｔＲＮＡ（アミノアシルｔＲＮＡ）を調製すること。その後、前記調製されたアミノアシルｔＲＮＡを蛋白質合成系に添加して蛋白質合成を行うこと。なおこのとき、前記蛋白質合成系にアミノアシルｔＲＮＡに加えて、さらにＹアナログと、アミノアシルｔＲＮＡ合成酵素変異体とを加えてもよい。また、第二実施形態の非天然蛋白質の製造方法も全く同様に製造され得る。
【００５３】
さらに、前記実施形態より把握できる技術的思想について以下に記載する。
・ 非天然蛋白質の製造方法により製造された非天然蛋白質であって、蛋白質構成アミノ酸としてチロシンアナログを備えていることを特徴とする非天然蛋白質。請求項２に記載の非天然蛋白質の製造方法により製造された非天然蛋白質であって、蛋白質構成アミノ酸としてチロシンアナログ修飾体を備えていることを特徴とする非天然蛋白質。
【００５４】
・ 非天然蛋白質の製造方法に用いられる非天然蛋白質の製造用のキットであって、チロシンアナログと、そのチロシンアナログをサプレッサーｔＲＮＡに結合させるアミノアシルｔＲＮＡ合成酵素変異体とを備えていることを特徴とするキット。このように構成した場合、生理活性の低下を抑えつつ、蛋白質構成アミノ酸として非天然アミノ酸を高効率で部位特異的に組込むことが容易である。
【００５５】
・ 非天然蛋白質の固定化方法に用いられる非天然蛋白質の固定化用のキットであって、チロシンアナログと、そのチロシンアナログをサプレッサーｔＲＮＡに結合させるアミノアシルｔＲＮＡ合成酵素変異体と、固定化担体とを備えていることを特徴とするキット。このように構成した場合、生理活性の低下を抑えつつ、部位特異的に非天然蛋白質を固定化することが容易である。
【００５６】
【発明の効果】
以上詳述したように、この発明によれば、次のような効果を奏する。
請求項１から請求項３に記載の発明の非天然蛋白質の製造方法によれば、生理活性の低下を抑えつつ、蛋白質構成アミノ酸として非天然アミノ酸を高効率で部位特異的に組込むことが容易である。請求項４に記載の発明の非天然蛋白質の固定化方法によれば、生理活性の低下を抑えつつ、部位特異的に非天然蛋白質を固定化することが容易である。
【００５８】
【配列表】

【図面の簡単な説明】
【図１】 第一実施形態の後修飾工程における反応の一例を示す模式図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a non-natural protein, which produces a non-natural protein in which a non-natural amino acid having a side chain that does not exist in 20 kinds of L-α-amino acids universally used by living organisms is site-specifically incorporated. , And Method for immobilizing non-natural proteins , And a kit for producing the non-natural protein.
[0002]
[Prior art]
Conventionally, as a method for producing this kind of non-natural protein, for example, a non-natural amino acid-incorporated protein, its production method and a reagent kit are known (see Patent Document 1). This production method uses an isoaccepting tRNA corresponding to a specific amino acid in a cell-free protein synthesis system in which only a part of the tRNA is bound to a non-natural amino acid and a protein-constituting amino acid. Protein synthesis is performed to incorporate unnatural amino acids into specific codon groups. The non-natural amino acid may be a. A substitution of an atom in a natural amino acid with a stable isotope, b. A substitution of an atom in a natural amino acid with a radioisotope, c. A side chain optical isomer of a natural amino acid, d. A substituent introduced into the side chain of a natural amino acid, e. Examples include those in which the side chain of a natural amino acid is substituted to change hydrophobicity, reactivity, fluorescence, charge state, molecular size, hydrogen bonding ability, and the like.
[0003]
On the other hand, a selenomethionine-substituted protein produced by, for example, a method disclosed in Non-Patent Document 1 has been conventionally used for X-ray crystal structure analysis of proteins. When producing this selenomethionine-substituted protein, first prepare a plasmid into which the methionine-requiring strain of E. coli and the gene of the target protein have been inserted, and then use an agarose gel plate containing antibiotics. Isolate a single colony expressing the protein of interest. Next, the isolated transformant is cultured in a medium containing selenomethionine instead of methionine to express and purify the target protein.
[0004]
[Patent Document 1]
Japanese Patent No. 3317983
[Non-Patent Document 1]
Toshiyuki Shimizu, Kengo Okada, Toshio Hakoshima, Chapter 17 X-ray crystal structure analysis Expression and preparation of selenomethionine-substituted protein, “Basic Biochemical Experimental Method 3 Protein I. Detection and Structural Analysis”, Tokyo Chemical Co., Ltd., First edition, first printing February 15, 2001, p. 189-190.
[0005]
[Problems to be solved by the invention]
However, in the conventional method for producing an unnatural amino acid-incorporating protein, the type, nature, size, etc. of the substituents d and e have not been carefully selected. However, it was not practical. Furthermore, in this production method, it was practically impossible to incorporate a non-natural amino acid into which a huge substituent that would become a steric hindrance in binding to a ribosome was introduced.
[0006]
On the other hand, when producing the conventional selenomethionine-substituted protein, site-specific substitution can be performed because selenomethionine is incorporated and substituted at all positions where the target protein methionine is to be incorporated. There wasn't. For this reason, in the protein produced by this production method, if the number of substitutions of selenomethionine is too large, the solubility is significantly reduced, making purification and crystallization difficult, and conversely, as a constituent amino acid in the target protein. When methionine was not included, various inconveniences occurred such that it was not applicable as it was.
[0007]
The present invention has been made paying attention to the problems existing in the prior art as described above. An object of the present invention is to provide a method for producing a non-natural protein that can easily incorporate a non-natural amino acid as a protein-constituting amino acid with high efficiency and site-specificity while suppressing a decrease in physiological activity. Another object is to provide a non-natural protein in which a non-natural amino acid is incorporated in a site-specific manner as a protein-constituting amino acid and it is easy to suppress a decrease in physiological activity. Another object is to provide a method for immobilizing a non-natural protein that can easily immobilize a non-natural protein in a site-specific manner while suppressing a decrease in physiological activity. On the other hand, another object of the present invention is to provide a non-natural protein that can be easily used for X-ray structural analysis, a production method thereof, and a kit for production.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, the method for producing a non-natural protein of the invention according to claim 1 is a method for producing a non-natural protein in which a non-natural amino acid is incorporated in a site-specific manner. A protein synthesis step in which a template is added to a protein synthesis system containing a suppressor tRNA and an aminoacyl tRNA synthetase mutant that binds the tyrosine analog to the suppressor tRNA. And post-modification step after the protein synthesis step Wherein the tyrosine analog is azidotyrosine, acetyltyrosine, aminotyrosine or dopa, and the template is a polynucleotide in which a stop codon recognized by the suppressor tRNA is arranged in the translation region. In the post-modification step, the tyrosine analog is modified with a fluorescent tyrosine analog by modifying the azide group, acetyl group, amino group or hydroxyl group of the side chain of the tyrosine analog and binding a modification containing a fluorescent substance. Process It is characterized by that.
[0009]
The method for producing a non-natural protein according to claim 2 is the method according to claim 1, wherein The tyrosine analog is azidotyrosine, and the modified product is a fluorescent substance and a triarylphosphine derivative bonded to the fluorescent substance. In Thus, the fluorescent substance and the triarylphosphine derivative are combined, and the combined modification product is combined with the azide group. It is characterized by this.
[0010]
The method for producing a non-natural protein according to claim 3 is the method according to claim 1 or 2, wherein the aminoacyl-tRNA synthetase mutant is used as the mutant. yeast( Saccharomyces cerevisiae ) And the suppressor tRNA is yeast( Saccharomyces cerevisiae ) Nonsense suppressor tRNA ^Tyr It is characterized by that.
[0011]
Contract Claim 4 The method for immobilizing a non-natural protein of the invention described in A method for producing a non-natural protein in which a non-natural amino acid is incorporated in a site-specific manner, and comprising a tyrosine analog, a suppressor tRNA, and an aminoacyl-tRNA synthetase variant that binds the tyrosine analog to a suppressor tRNA The synthesis system includes a protein synthesis step in which a template is added to synthesize the protein, and the tyrosine analog is azidotyrosine, acetyltyrosine, aminotyrosine or dopa, and the template has a stop codon recognized by the suppressor tRNA in the translation region. Characterized by being a polynucleotide A method for immobilizing a non-natural protein produced by a method for producing a non-natural protein on an immobilization carrier comprising a support and a reactive group bonded to the support, the azide of the side chain of the tyrosine analog A group, an acetyl group, an amino group or a hydroxyl group is bonded to the reactive group.
[0012]
Claim 5 The kit of the invention described in claim 1 is a kit for producing a non-natural protein used in the method for producing a non-natural protein according to claim 1, wherein the tyrosine analog, the aminoacyl-tRNA synthetase mutant, And a modified product containing a fluorescent substance.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
(First embodiment)
Hereinafter, a first embodiment of the present invention will be described in detail.
[0016]
The unnatural protein of the first embodiment is a polypeptide in which an unnatural amino acid is incorporated in a site-specific manner. The unnatural amino acid is an amino acid having a side chain that does not exist in 20 kinds of standard amino acids (L-α-amino acids) universally used by living organisms, and is a tyrosine analog (Y analog) or a modified form thereof (tyrosine analog). Modified, Y analog modified). This non-natural protein is classified into two types: a first non-natural protein in which the Y analog is incorporated, or a second non-natural protein in which the modified Y analog is incorporated.
[0017]
As the Y analog, azidotyrosine, acetyl tyrosine, amino tyrosine, or dopa (DOPA; 3,4-dihydroxyphenylalanine) is used. These Y analogs have a structure shown in Chemical Formula 1 or Chemical Formula 2 below.
[0018]
[Chemical 1]

However, R1 is an azide group, an acetyl group, an amino group, or a hydroxyl group.
[0019]
[Chemical 2]

However, R2 is an azido group, an acetyl group, or an amino group.
[0020]
That is, the Y analog represented by Chemical Formula 1 has an azido group (—N) as a substituent R1 at the 3 (meth) position of the phenolic aromatic ring constituting the side chain of tyrosine. _Three ), Acetyl group (-COCH _Three ), Amino group (-NH ₂ ) Or a hydroxyl group (—OH). The Y analog shown in Chemical Formula 2 has an azide group, acetyl group or amino group as a substituent R2 at the 2 (ortho) position of the phenolic aromatic ring constituting the side chain of tyrosine. These Y analog substituents may be bonded to either the meta position or the ortho position, but are located at the end of the side chain and the reaction to form a Y analog modified product is likely to proceed. It is preferably bonded to the meta position. These Y analogs may be either L-type or D-type optical isomers, but are preferably L-type because of high protein synthesis efficiency.
[0021]
The modified Y analog is an organic compound in which a modified product is bonded to the side chain of the Y analog (position 2 or position 3 of the phenolic aromatic ring). The modified product includes a modifying group and a reactive group bonded to the modifying group. Examples of the reactive group include orthoalkoxycarbonylaryldiarylphosphine (triarylphosphine derivative), hydrazine, succimidyl ester, isothiocyanate salt, sulfonyl chloride, Examples include aldehyde, carboxylic acid, acid halide, or acid anhydride thereof. The modifying group is not particularly limited. However, since it is easy to expand practical applications, fluorescent substances, hydrophobic macromolecules, hydrophilic substances, substances having electric charge, substances having hydrogen bonding ability, chemical reactivity Substances having a bioactivity, physiologically active substances (enzymes, etc.), biological constituents (proteins, sugar chains, polynucleotides, fatty acids, etc.), biotin, antibodies or parts thereof are preferably used. Moreover, you may use the functional support | carrier used for drug delivery systems (DDS), such as polyethyleneglycol, as a modification group.
[0022]
This modified Y analog is obtained by chemically or biochemically coupling (covalently bonding) a substituent of the Y analog and a reactive group of the modified product.
As a combination of the substituent and the reactive group, when the substituent is an azide group, the reactive group is a triarylphosphine derivative, and a reaction (Staudinger ligation reaction) schematically shown in FIG. 1 is performed. Is called. In addition, when the substituent is an acetyl group, the reactive group is hydrazine, and when the substituent is an amino group, the reactive group is succinimide ester, isothiocyanate, sulfonyl chloride, aldehyde, carboxylic acid or acid halide or It becomes the acid anhydride. The reaction between these substituents and reactive groups is a chemical reaction. In addition, when the Y analog is dopa, the hydroxyl groups at the 3rd and 4th positions of the phenolic aromatic ring are combined with an amino group as a reactive group using an enzyme reaction by DOPA oxidase (biological organism). By chemical reaction), the modified product is bonded to form a Y analog modified product. The reaction for modifying the substituent of these Y analogs can be carried out under physiological conditions (for example, conditions under which general bacteria such as Escherichia coli and yeast or single cell organisms can survive).
[0023]
The first method for producing a non-natural protein according to the embodiment includes a protein synthesis step in which a protein is synthesized by adding a template to a protein synthesis system containing a Y analog, a suppressor tRNA, and an aminoacyl tRNA synthetase mutant. It is. The suppressor tRNA is a nonsense suppressor tRNA or a missense suppressor tRNA that reads a stop codon (amber, ocher or opal) on mRNA as a codon corresponding to a certain amino acid.
[0024]
The aminoacyl-tRNA synthetase mutant is an enzyme that specifically binds the Y analog to the suppressor tRNA. The aminoacyl-tRNA synthetase mutant is not particularly limited as long as it uses the Y analog as a substrate. However, since the substrate specificity for a standard amino acid tends to be low, a wild-type tyrosyl-tRNA synthetase is used. A modified tyrosyl-tRNA synthetase mutant (TyrRS mutant) is most preferably used. Examples of the wild-type tyrosyl-tRNA synthetase include yeast (Saccharomyces cerevisiae) tyrosyl-tRNA synthetase having the amino acid sequence represented by SEQ ID NO: 1.
[0025]
As a mutant of this yeast tyrosyl tRNA synthetase, ie, a yeast TyrRS mutant, a Y43G tyrosyl tRNA synthetase mutant (Y43G TyrRS) in which the 43rd Tyr of the amino acid sequence represented by SEQ ID NO: 1 is substituted with Gly, H81V tyrosyl-tRNA synthetase mutant (H81V TyrRS) in which the 81st His in the amino acid sequence represented by SEQ ID NO: 1 is substituted with Val, and the 186th Gln in the amino acid sequence represented by SEQ ID NO: 1 is substituted with Val Q186V tyrosyl tRNA synthetase mutant (Q186V TyrRS), Q186T tyrosyl tRNA synthetase mutant (Q186T TyrRS) in which the 186th Gln of the amino acid sequence represented by SEQ ID NO: 1 is replaced with Thr, or SEQ ID NO: 1 Q186I tyrosy in which the 186th Gln of the amino acid sequence represented by And a tRNA synthetase mutant (Q186I TyrRS). Moreover, the variant which consists of the 1st -364th amino acid sequence of said Y43G TyrRS, H81V TyrRS, Q186V TyrRS, Q186T TyrRS, or Q186I TyrRS is used from having the same enzyme activity.
[0026]
Furthermore, as this TyrRS mutant, it is preferable to use a TyrRS mutant in which a tag such as a histidine tag is bound to the C-terminus of each TyrRS mutant. The Y43G TyrRS is disclosed in S. Ohno et.al., Changing the Amino Acid Specificity of Yeast Tyrosyl-tRNA Synthetase by Genetic Engineering, J. Biochem. 130, 417-423 (2001). Each of the yeast TyrRS mutants is a yeast nonsense suppressor tRNA. ^Tyr (TRNA ^Tyr ) (Amber, ocher or opal).
[0027]
The template is a polynucleotide in which a stop codon recognized by the suppressor tRNA is artificially arranged in a translation region by site-directed mutagenesis or the like, and is used in the form of DNA or mRNA. Examples of the DNA include double-stranded DNA containing a sequence essential for transcription, and it is most convenient to introduce the DNA into various vectors and plasmids. The stop codon located at the 3 ′ end of the translation region is a codon having a base sequence different from the stop codon recognized by the suppressor tRNA.
[0028]
The protein synthesis system is a system for synthesizing the protein encoded by the template, and examples thereof include an in vitro protein synthesis system and an in vivo protein synthesis system. The aminoacyl-tRNA synthetase mutant contained in this protein synthesis system and the suppressor tRNA as a substrate of the mutant are derived from a different species of organism from the components involved in the translation process of the protein synthesis system. Preferably, one is from a eukaryotic cell and the other is from a prokaryotic cell. At this time, it becomes much easier to incorporate the Y analog into the first non-natural protein appropriately and site-specifically.
[0029]
Examples of the in vitro protein synthesis system include an in vitro translation system that performs translation from mRNA as a template, or an in vitro transcription and translation system that performs transcription and translation from DNA as a template. These in vitro protein synthesis systems are constructed by artificially mixing transcription or components necessary for transcription and translation, cell-free protein synthesis systems consisting of cell extracts, or by mixing both Is used. The cell-free protein synthesis system is obtained by enucleating mainly prokaryotic cells such as E. coli, eukaryotic cells such as yeast, plant cells such as protoplasts, insect cells, and mammalian cells, and is used for transcription and translation. Various necessary components are highly contained. Since this in vitro protein synthesis system has been widely recognized as useful, one containing E. coli S30 extract or wheat germ extract, haemolysis system of rabbit reticulocytes, Chinese hamster ovary cells (CHO), baculovirus Vector protein expression systems, Xenopus oocytes and the like are preferably used. In addition, E. coli cell extracts from which factors (such as nucleolytic enzymes, proteolytic enzymes, dissociation factors, oxidative / reductive enzymes, etc.) that negatively affect the introduction of unnatural amino acids have been destroyed (deleted) or removed by immunoprecipitation Preferably used.
[0030]
The Y analog contained in the in vitro protein synthesis system is preferably more than the content of each standard amino acid in the protein synthesis system, more preferably 1 to 100 times the content, more preferably 5 It is good that the amount is 10 times. If the Y analog is less than 1 times the content, the Y analog cannot be incorporated into the non-natural protein with high efficiency. Conversely, if it exceeds 100 times, the order of the protein synthesis system is disturbed. It becomes easy to fail. In addition, the suppressor tRNA contained in the in vitro protein synthesis system is preferably the same as the content of each tRNA in the protein synthesis system, more preferably 0.01 to 10 times the above content, The amount is preferably 0.5 to 2 times. If the suppressor tRNA is less than 0.01 times the content, the Y analog cannot be incorporated into the non-natural protein with high efficiency. Conversely, if the suppressor tRNA exceeds 10 times the content, the order of the protein synthesis system Disrupted and prone to bankruptcy. In addition, the aminoacyl-tRNA synthetase variant contained in the in vitro protein synthesis system is preferably more than the content of each aminoacyl-tRNA synthetase (wild type) in the protein synthesis system, and more preferably 0.05 to 0.5 mg / ml, more preferably 0.1 to 0.2 mg / ml. If the aminoacyl-tRNA synthetase variant is less than 0.05 mg / ml of the above content, the Y analog cannot be incorporated into the non-natural protein with high efficiency, and conversely if it exceeds 0.5 mg / ml. The order of the protein synthesis system is disturbed and it tends to break down.
[0031]
As an in vivo protein synthesis system, a cell (transformed cell) transformed so that the template as an exogenous gene and an aminoacyl-tRNA synthetase mutant can be expressed, or an individual organism containing the cell is used. As the transformed cells, living cells that have not been enucleated are used, and prokaryotic cells such as E. coli, eukaryotic cells such as yeast, plant cells, insect cells, mammalian cells, and the like are preferably used. When a suppressor tRNA as a substrate of the aminoacyl tRNA synthetase mutant is not endogenous to the transformed cell, the suppressor tRNA must also be transformed into the same cell as a foreign gene. In this in vivo protein synthesis system, by feeding a Y analog as a diet, the protein synthesis system in a transformed cell in which the foreign gene is expressed continuously and produces a large amount of non-natural protein.
[0032]
On the other hand, as the Y analog used in the first method for producing a non-natural protein, azidotyrosine or dopa is preferably used because it has a substituent not usually contained in the protein synthesis system. In addition, since the second non-natural protein can be produced using a highly specific enzyme reaction, dopa is particularly preferably used.
[0033]
According to the second method for producing a non-natural protein of the embodiment, after the protein synthesis step in the first method for producing a non-natural protein is performed, a post-modification step is further performed. This post-modification step is a step of modifying the azide group, acetyl group, amino group or hydroxyl group of the side chain of the Y analog to make a Y analog modified product, and the chemical reaction or biochemical reaction in the Y analog modified product. Reaction takes place. In the second method for producing a non-natural protein, it is preferable to perform an operation of removing an unreacted Y analog not involved in protein synthesis between the protein synthesis step and the post-modification step.
[0034]
In the method for immobilizing a non-natural protein according to an embodiment, the first non-natural protein is immobilized on an immobilization carrier. The immobilization carrier includes a support made of a solid insoluble in water or an organic solvent, and a reactive group bonded to the support. As the reactive group, one that reacts with a substituent of the modified Y analog is the same as the reactive group. In this immobilization method, the first non-natural protein is immobilized on the immobilization carrier by performing the same reaction as the chemical reaction or biochemical reaction in the Y analog modified product. The immobilization carrier on which the non-natural protein is immobilized can be used for, for example, affinity chromatography, protein chip, bioreactor and the like. In addition, extracellular matrix such as collagen, proteoglycan, fibronectin, elastin, and hyaluronic acid, and cell adhesion molecules such as cadherin family, immunoglobulin superfamily, integrin family, selectin family, link protein family, and sialomucin family are immobilized. It can also be used for implant materials and artificial materials for regenerative medicine.
[0035]
The effects exhibited by the first embodiment will be described below.
-The 1st non-natural protein of 1st embodiment is a site-specific integration of Y analog as a protein constituent amino acid. The second non-natural protein of the first embodiment is one in which a modified Y analog is site-specifically incorporated as a protein-constituting amino acid. That is, these non-natural proteins are artificially imparted with properties different from those of natural proteins because non-natural amino acids are incorporated site-specifically as protein-constituting amino acids.
[0036]
For this reason, since these non-natural proteins are clearly distinguished in molecular structure from natural proteins, it is easy to examine their localization and behavior in vivo. In particular, in the first non-natural protein, since the size of the Y analog is hardly different from that of the standard amino acid, there is very little adverse effect on the living organism, and a post-modification step is performed on the organism in situ ( In situ visualization is extremely easy. It is also easy to label (visualize) after electrophoresis. In addition, these non-natural proteins have site-specific incorporation of non-natural amino acids, so they can be used to identify protein constituent amino acids that are involved in the physiological activity of natural proteins, and to be used for structural analysis of proteins. And can be used to develop information processing analysis technology. In addition, research or industrial application of affinity chromatography carriers, protein chips and their analysis equipment, bioreactors, nanotechnology, electronic equipment, information analysis, precision measurement equipment, etc. is easy. It can also be applied to medical-related fields such as the development of innovative drug materials and biomarkers that utilize genomic information. In particular, the first non-natural protein is obtained by a biological production method using a protein synthesis system, and the second non-natural protein is subjected to a chemical reaction between a substituent and a reactive group under physiological conditions. Therefore, it is easy to stop the change of the physiological activity and the change of the three-dimensional structure extremely easily, and it is also easy to artificially change it.
[0037]
-Y analog is used in the manufacturing method of the 1st non-natural protein of 1st embodiment. The azido group, acetyl group, amino group, or hydroxyl group of these Y analogs all clarifies the difference in molecular structure with respect to the standard amino acid, and all have a small size that hardly causes steric hindrance. For this reason, all these Y analogs have a size that does not cause steric hindrance when the reaction of aminoacyl tRNA binding to the aminoacyl tRNA binding site (A site) on the ribosome is performed. Proceeds almost as quickly as the case. Furthermore, since these Y analogs are incorporated into non-natural proteins by a physiological reaction using a protein synthesis system according to the central dogma, the decrease in the physiological activity of the protein is remarkably effectively suppressed. Yes. In addition, since the genetic code consisting of triplets is used, the translation frame of a polynucleotide encoding a known natural protein does not need to be significantly modified by site-directed mutagenesis etc., and can be produced very easily. Is possible.
[0038]
In the first embodiment, the second non-natural protein production method comprises producing a first non-natural protein by performing a protein synthesis step, and then mixing the protein and a modified product to react under physiological conditions. A modified product is bound to the protein by performing a post-modification step (chemical reaction, enzyme reaction, etc.). In this production method, since both the protein synthesis step and the post-modification step are performed under physiological conditions, a decrease in the physiological activity of the second non-natural protein can be extremely effectively suppressed. Furthermore, the reaction between the substituent and the reactive group in the post-modification step is a reaction that proceeds remarkably quickly and efficiently. On the other hand, since the immobilization method of the non-natural protein of the embodiment uses almost the same reaction as that in the post-modification step, the same effect can be achieved.
[0039]
(Second embodiment)
Hereinafter, a second embodiment of the present invention will be described with reference to the first embodiment.
The non-natural protein of the second embodiment is a polypeptide in which bromotyrosine as a Y analog is incorporated in a site-specific manner. This non-natural protein is suitably used for X-ray crystal structure analysis of the non-natural protein because a bromo group (-Br) having a property of suitably scattering X-rays is incorporated in a site-specific manner. . Further, in this non-natural protein, since the bromo group is a substituent having very little steric hindrance, it is difficult for a difference to occur between the three-dimensional structure (tertiary structure and quaternary structure) of the non-natural protein and that of the natural protein. A very suitable structural analysis can be performed.
[0040]
The bromotyrosine is a Y analog having a bromo group as a substituent at the 3 (meth) -position or 2 (ortho) -position of the phenolic aromatic ring constituting the side chain of tyrosine. Examples include those in which the substituent R1 of the Y analog is a bromo group, or the substituent R2 of the Y analog shown in Chemical Formula 2 is a bromo group. The bromo group of this bromotyrosine may be bonded to either the meta position or the ortho position. The bromotyrosine may be either L-type or D-type optical isomer, but L-type is preferable because of high protein synthesis efficiency.
[0041]
The method for producing a non-natural protein of the second embodiment is carried out in the same manner as the method for producing a first non-natural protein of the first embodiment.
The kit for producing a non-natural protein as the kit of the second embodiment contains at least bromotyrosine and an aminoacyl-tRNA synthetase mutant that binds the bromotyrosine to a suppressor tRNA, and further contains a suppressor tRNA. Is more preferable. The production kit preferably further contains a protein synthesis system. At this time, the suppressor tRNA and aminoacyl-tRNA synthetase mutant are preferably derived from a different species of organism from the component involved in the translation process of the protein synthesis system, one of which is derived from a eukaryotic cell. It is even more preferred that the other is derived from prokaryotic cells. In addition, the production kit may contain a gene kit for performing site-specific mutation.
[0042]
The effects exhibited by the second embodiment will be described below.
-The non-natural protein of the second embodiment is one in which bromotyrosine is incorporated site-specifically as a protein-constituting amino acid. For this reason, since the non-natural protein has almost the same bromotyrosine size as the standard amino acid, the tertiary structure of the non-natural protein is hardly different from that of the natural protein. Therefore, it is preferably used for examining the three-dimensional structure in the X-ray crystal structure analysis, and in that case, it is very excellent in the X-ray scattering characteristics, which is preferable. Furthermore, since bromotyrosine is incorporated in a site-specific manner, there is almost no occurrence of various problems as in the case of the conventional selenomethionine-substituted protein.
[0043]
In the method for producing a non-natural protein of the second embodiment, bromotyrosine is used as the Y analog. Since the bromo group of this Y analog has a small size that hardly causes steric hindrance, it has exactly the same effect as the first method for producing a non-natural protein of the first embodiment.
[0044]
【Example】
Hereinafter, examples and comparative examples embodying the embodiment will be described.
<Preparation of in vitro protein synthesis system (E. coli S30 extract)>
A single colony of Escherichia coli Q13 strain (distributed from the National Institute of Genetics) grown on an LB (Luria-Bertani) plate was inoculated into 100 ml of LB medium, and cultured overnight (preculture) at 37 ° C. overnight. Next, the culture solution after the pre-culture is added to 100 times amount of 2 × YT (Trypton Pepton, Yeast Extract) medium (manufactured by DIFCO), and turbidity at 600 nm (OD ₆₀₀ ) Was shake-cultured (main culture) at 37 ° C. until it became 0.3 to 0.4. Subsequently, the culture solution after the main culture was ice-cooled and immediately centrifuged to collect the cells, and then the cells were mixed with Hepes buffer (Hepes-KOH (pH 7.5) 20 mM), magnesium acetate (10 mM). ), Ammonium acetate (20 mM), and 2-mercaptoethanol (10 mM) in 40 ml of a solution to obtain a cell suspension. Lysozyme (manufactured by Seikagaku Corporation) was added to the cell suspension to a concentration of 2 mg / ml, and the mixture was allowed to stand on ice for about 10 minutes. Subsequently, the cell suspension was centrifuged to obtain a precipitate, the precipitate (cell) was weighed, and the same amount of S30 buffer solution (1 ml of S30 buffer per 1 g of cell) was obtained. To obtain an S30 suspension. The S30 suspension was crushed and centrifuged to obtain a supernatant. This supernatant is an Escherichia coli S30 extract as an in vitro protein synthesis system, and can be subdivided and stored for a long time in liquid nitrogen.
[0045]
<Manufacture of non-natural protein>
Hepes buffer (Hepes-KOH (pH7.6) 50 mM), magnesium acetate (7.7 mM), ammonium acetate (27.5 mM), dithiothreitol (DTT 1.7 mM), nucleotide triphosphate (ATP 1.25 mM, GTP 0.83 mM) , UTP 0.83 mM, CTP 0.83 mM), creatine phosphate (80 mM), creatine kinase (0.21 mg / ml), T7 RNA polymerase (0.1 mg / ml isolated by the present inventors), E. coli Q13 strain tRNA mix (present invention) Isolated 3.4A _260unit / ml), L-tyrosine (80 μM), 19 standard amino acids other than L-tyrosine (each 160 μM), folinic acid (50 μM), potassium acetate (200 mM), polyethylene glycol (wt.8000 4%), 3- Azido-L-tyrosine (synthesized by the inventors: 500 μM), yeast Y43G TyrRS (produced by the inventors: 0.17 mg / ml), yeast suppressor tRNA ^Tyr (CUA) (isolated by the present inventors 0.1A _260unit / ml), template DNA (20 μg / ml), E. coli S30 extract (30% (v / v)) and deionized water are placed in a test tube and mixed to the final concentration indicated in parentheses. And then incubated at 30 ° C. for 1 hour.
[0046]
In Comparative Example 1, a wild-type E. coli dihydrofolate reductase [EC: 1.5.1.3]; DHFR) gene, that is, a double-stranded DNA consisting of the base sequence represented by SEQ ID NO: 2 is incorporated as a template DNA. The resulting plasmid vector pET 21-a (+) (NOVAGEN) was used. In Comparative Example 2, the plasmid vector pET 21-a (+) was used as the template DNA. In Example 1, as a template DNA, a gene in which the codon corresponding to Asn at position 37 of E. coli DHFR was changed to an amber codon by site-directed mutagenesis, that is, the 109th to 111th positions of the base sequence represented by SEQ ID NO: 2 were used. Plasmid vector pET 21-a (+) in which double-stranded DNA modified in tag was incorporated was used. In Example 2, as a template DNA, a codon corresponding to Tyr at position 128 of E. coli DHFR was changed to an amber codon by site-directed mutagenesis, that is, the base sequence 382 to 384 represented by SEQ ID NO: 2. A plasmid vector pET 21-a (+) in which a double-stranded DNA modified with the second tag was used was used.
[0047]
Next, 3-azide-L-tyrosine, which was not involved in protein synthesis, was removed from the reaction solution after the incubation with a Ni-NTA spin column (Qiagen, eluent was a phosphate buffer solution at pH 7.4). After purification, the fluorescently labeled modified product was added to a final concentration of 250 μM and incubated at 37 ° C. for 1 hour. Subsequently, Examples 1, 2 and Comparative Examples 1 and 2 and wild-type Escherichia coli DHFR isolated in advance were subjected to SDS gel electrophoresis, followed by Coomassie Brilliant Blue (CBB) staining and fluorescence staining. The modified product is a triarylphosphine derivative synthesized by the present inventors and has a structure as shown in FIG.
[0048]
As a result, no fluorescence was detected in Comparative Examples 1 and 2, but in Examples 1 and 2 produced using the modified template DNA, the position of the CBB-stained band having the same molecular weight as that of wild-type Escherichia coli DHFR. A fluorescent band was detected. Moreover, although data are not shown, DHFR activity of almost the same level as that of wild-type Escherichia coli DHFR was confirmed in the unnatural proteins of Examples 1 and 2.
[0049]
<Immobilization of non-natural proteins>
A triallyl phosphine derivative (5 mg / ml dimethyl sulfoxide solution) was dispensed into each hole (well) of an amino coating plate (manufactured by Sumitomo Bakelite Co., Ltd.) and incubated at 37 ° C. for 2 hours for adsorption. After each well was washed with a phosphate buffer (pH 7.4), Examples 1, 2 and Comparative Examples 1 and 2 and a phosphate buffer (control) were dispensed into each well and incubated at 37 ° C. for 6 hours. Incubated. Next, each well was washed with a phosphate buffer (pH 7.4), and then methotrexate labeled with Texas Red was dispensed into each well and incubated at 37 ° C. for 2 hours. Finally, each well was washed with a phosphate buffer (pH 7.4) and then analyzed with an imaging analyzer (manufactured by Amersham Pharmacia Biotech). The color of Texas Red was clearly confirmed. On the other hand, it was not confirmed in the wells into which Comparative Examples 1 and 2 and phosphate buffer were dispensed.
[0050]
<Investigation of substrate specificity of aminoacyl tRNA synthetase mutant>
Substrate specificity for various Y analogs was confirmed by polyacrylamide gel electrophoresis under acidic conditions for five types of yeast TyrRS mutants, Y43G TyrRS, H81V TyrRS, Q186V TyrRS, Q186T TyrRS, and Q186I TyrRS. The electrophoresis was performed according to the method of Nucleic Acids Symposium Series No. 35, 285-286 (1996) (Oxford University Press, ISSN 0261 3166). The results are shown in Table 1. In Table 1, when it was actually confirmed that the yeast TyrRS recognized the Y analog, it was indicated as “◎”. In addition, although it is not directly confirmed that yeast TyrRS recognized the Y analog, from the test results of wild type yeast tyrosyl-tRNA synthetase (yeast TyrRS), etc., When the possibility of recognizing is sufficient, it is indicated by Δ, when the possibility of recognizing is very low or not recognized, x is indicated, and when it is unclear whether it is recognized or not, ND is indicated.
[0051]
[Table 1]

As a result, although all of these five types of yeast TyrRS mutants have not been confirmed, they are generally used for the production of the non-natural protein of each of the above embodiments although there are some differences in substrate specificity. It was confirmed that it was possible. Furthermore, these five types of yeast TyrRS mutants have a substituent at the meta position (position 3) as shown in Table 1, but the wild type yeast TyrRS has a substituent at the ortho position (position 2). There is a high possibility of recognizing in the same way from the fact that what is possessed is recognized as a substrate. On the other hand, although Y43G TyrRS and H81V TyrRS do not recognize D-tyrosine, the possibility of recognizing a D-type Y analog is extremely low, but D-dopa is highly likely to be recognized. Q186V TyrRS, Q186T TyrRS and Q186I TyrRS are highly likely to recognize D-tyrosine as a substrate, but D-dopa is very unlikely to be a substrate.
[0052]
In addition, the said embodiment can also be changed and actualized as follows.
In the first method for producing a non-natural protein according to the first embodiment, first, a Y analog is aminoacylated by first enzymatically reacting a Y analog, a suppressor tRNA, and an aminoacyl tRNA synthetase mutant. Preparing tRNA (aminoacyl tRNA). Thereafter, the prepared aminoacyl-tRNA is added to a protein synthesis system for protein synthesis. At this time, in addition to the aminoacyl tRNA, a Y analog and an aminoacyl tRNA synthetase mutant may be added to the protein synthesis system. Moreover, the manufacturing method of the non-natural protein of the second embodiment can be manufactured in exactly the same manner.
[0053]
Further, the technical idea that can be grasped from the embodiment will be described below.
・ Non A non-natural protein produced by a method for producing a natural protein, comprising a tyrosine analog as a protein-constituting amino acid. A non-natural protein produced by the method for producing a non-natural protein according to claim 2, comprising a tyrosine analog modified product as a protein-constituting amino acid.
[0054]
・ Non A kit for producing a non-natural protein used in a method for producing a natural protein, comprising a tyrosine analog and an aminoacyl-tRNA synthetase mutant that binds the tyrosine analog to a suppressor tRNA. . When configured in this way, it is easy to incorporate a non-natural amino acid as a protein-constituting amino acid with high efficiency and site-specificity while suppressing a decrease in physiological activity.
[0055]
・ Non A kit for immobilizing a non-natural protein used in a method for immobilizing a natural protein, comprising a tyrosine analog, an aminoacyl-tRNA synthetase mutant that binds the tyrosine analog to a suppressor tRNA, and an immobilization carrier Kit characterized by being. When comprised in this way, it is easy to fix | immobilize a non-natural protein site-specifically, suppressing the fall of physiological activity.
[0056]
【The invention's effect】
As described in detail above, the present invention has the following effects.
According to the method for producing an unnatural protein of the invention described in claims 1 to 3, it is easy to incorporate a non-natural amino acid as a protein-constituting amino acid with high efficiency and site-specificity while suppressing a decrease in physiological activity. is there . Contract Claim 4 According to the method for immobilizing a non-natural protein of the invention described in (2), it is easy to immobilize a non-natural protein in a site-specific manner while suppressing a decrease in physiological activity.
[0058]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of a reaction in a post-modification step of the first embodiment.

Claims (5)

A method for producing a non-natural protein in which a non-natural amino acid is incorporated in a site-specific manner,
A protein synthesis step comprising adding a template to a protein synthesis system containing a tyrosine analog, a suppressor tRNA, and an aminoacyl-tRNA synthetase mutant that binds the tyrosine analog to a suppressor tRNA, and a protein synthesis step after the protein synthesis step Including a modification step,
The tyrosine analog is azidotyrosine, acetyltyrosine, aminotyrosine or dopa;
The template is a polynucleotide in which a stop codon recognized by the suppressor tRNA is arranged in the translation region,
In the post-modification step, the tyrosine analog is modified to be a fluorescent tyrosine analog by modifying the azide group, acetyl group, amino group or hydroxyl group of the side chain of the tyrosine analog and binding a modification containing a fluorescent substance. A method for producing a non-natural protein, which is a process.   The tyrosine analog is azidotyrosine, and the modified product is a fluorescent substance and a triarylphosphine derivative bonded to the fluorescent substance, the fluorescent substance and the triarylphosphine derivative are combined, and the combined modified product is The method for producing a non-natural protein according to claim 1, wherein the non-natural protein is bound to the azide group.   The aminoacyl-tRNA synthetase mutant is a tyrosyl-tRNA synthetase mutant of yeast (Saccharomyces cerevisiae), and the suppressor tRNA is a nonsense suppressor tRNATyr of yeast (Saccharomyces cerevisiae). 3. A method for producing a non-natural protein according to 2. A method for producing a non-natural protein in which a non-natural amino acid is incorporated in a site-specific manner, and comprising a tyrosine analog, a suppressor tRNA, and an aminoacyl-tRNA synthetase variant that binds the tyrosine analog to a suppressor tRNA The synthesis system includes a protein synthesis step in which a template is added to synthesize the protein, and the tyrosine analog is azidotyrosine, acetyltyrosine, aminotyrosine or dopa, and the template has a stop codon recognized by the suppressor tRNA in the translation region. In this method, the non-natural protein produced by the method for producing a non-natural protein , characterized by being a polynucleotide, is immobilized on an immobilization carrier comprising a support and a reactive group bound to the support. And
A method for immobilizing a non-natural protein, wherein the azide group, acetyl group, amino group or hydroxyl group of the side chain of the tyrosine analog is bonded to the reactive group. A kit for producing a non-natural protein used in the method for producing a non-natural protein according to claim 1 ,
A kit comprising the tyrosine analog, the aminoacyl-tRNA synthetase mutant, and a modified product containing the fluorescent substance.

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