JP6959251B2 - ナノポアシステムに有用な部位特異的バイオコンジュゲーション方法および組成物 - Google Patents
ナノポアシステムに有用な部位特異的バイオコンジュゲーション方法および組成物 Download PDFInfo
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- 238000000354 decomposition reaction Methods 0.000 description 1
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- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
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- 230000029226 lipidation Effects 0.000 description 1
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- 238000001819 mass spectrum Methods 0.000 description 1
- OVHQWOXKMOVDJP-UHFFFAOYSA-N melitin Chemical compound OC1C(O)C(O)C(C)OC1OC1C(O)C(OC=2C=C3C(C(C(OC4C(C(O)C(O)C(COC5C(C(O)C(O)C(CO)O5)O)O4)O)=C(C=4C=CC(O)=CC=4)O3)=O)=C(O)C=2)OC(C)C1O OVHQWOXKMOVDJP-UHFFFAOYSA-N 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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Description
(a)適切な反応条件下で、チオール基を含むタンパク質を、式(I)
の化合物と接触させ、それによって、式(II)
の修飾タンパク質を形成するステップと、
(b)式(ii)の修飾タンパク質を、式(iii)
の化合物と接触させ、それによって、構造式(IV)
(c)式(IV)の修飾タンパク質を、適切な条件下で、反応性基Bと共有結合を形成することが可能である反応性基Zを含む生体分子と接触させ、それによって、式(V)
のタンパク質−生体分子コンジュゲートを形成するステップ。
の修飾細孔形成性タンパク質を含む組成物を提供する。
のタンパク質−生体分子コンジュゲートを含む組成物を提供する。
の得られたタンパク質−生体分子コンジュゲートに関する。
の修飾細孔形成性タンパク質を含む中間体組成物を含む、タンパク質の、生体分子への部位選択的コンジュゲーションの方法において中間体として形成される化合物および組成物を提供する。
(a)適切な反応条件下で、チオール基を含むタンパク質を、式(I)
の修飾タンパク質を形成するステップと、
(b)式(ii)の修飾タンパク質を、式(iii)
の化合物と接触させ、それによって、構造式(IV)
(c)式(IV)の修飾タンパク質を、適切な条件下で、反応性基Bと共有結合を形成することが可能である反応性基Zを含む生体分子と接触させ、それによって、式(V)のタンパク質−生体分子コンジュゲートを形成するステップと
を含む。
[0032]定義
[0033]本明細書の説明において使用される技術用語および科学用語は、別段の定めがない限り、当業者に一般に理解される意味を有する。したがって、以下の用語は、以下の意味を有するものとする。
[0046]細孔形成性タンパク質、α−溶血素などのタンパク質と、DNAポリメラーゼなどの生体分子との間のコンジュゲートの調製に関する本明細書において開示する部位選択的コンジュゲーション方法は概して、リンカーおよびタンパク質または生体分子のいずれか上の基と反応する反応性基(または反応性部分)を含む試薬を要する。このコンジュゲーション方法は概して、下記ステップ(a)、(b)および(c)を含む:
(a)適切な反応条件下で、チオール基を含むタンパク質を、式(I)
(b)式(II)の修飾タンパク質を、式(III)
(c)式(IV)の修飾タンパク質を、適切な条件下で、反応性基Bと共有結合を形成することが可能である反応性基Zを含む生体分子と接触させ、それによって、式(V)のタンパク質−生体分子コンジュゲートを形成するステップ。
[0049]ステップ(a)は、タンパク質上のチオール基と、式(I)のクリック化学反応性基を含むリンカーとの共有結合的修飾を含み、式(II)の修飾タンパク質を生じる。このステップは、タンパク質が容易かつ効率的なクリック化学反応を介したさらなる修飾が可能であるように、タンパク質を本質的に修飾する。
[0059]一部の実施形態においては、式(I)の化合物は、表1に示すような式(Ia)または式(Ib)の化合物から選択される化合物を含む。
[0064]
[0066]
[0068]ステップ(b)は、式(II)の修飾タンパク質上のX基、および式(III)の試薬化合物上の同族クリック化学基Yのクリック化学反応を含む。このステップは、式(IV)および式(IVa)の中間体修飾タンパク質組成物を生じる(上記を参照のこと)。ステップ(c)において生体分子と最終的な部位選択的反応を起こすのは、式(IV)のこの中間体化合物上の反応性基Bである。
[0081]反応性基ZがN末端システイン残基を含むさらに特定の実施形態においては、式(III)の化合物は、表7に示すような式(IIIc)または式(IIId)の化合物を含み得る。
[0093]本開示の方法においてSpyCatcherタンパク質として有用な化膿連鎖球菌(Streptococcus pycogenes)フィブロネクチン結合タンパク質FbaBのCnaB2ドメインは、Liら、J.Mol.Biol.2014年1月23日;426(2):309〜317からの下記の144aaのアミノ酸配列を含み得る:
SYYHHHHHHDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDA(配列番号5)。
DYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI(配列番号6)。一部の実施形態においては、本開示の方法においてSpyCatcherタンパク質として有用なN末端CnaB2ドメイン配列断片は、Liら、J.Mol.Biol.2014年1月23日;426(2):309〜317からの下記の138aaのアミノ酸配列を含む:
SYYHHHHHHDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI(配列番号7)。
MHHHHHHHHSGDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGS(配列番号8)。
[0099]ステップ(c)は、構造式(IV)の修飾タンパク質の反応性基Bと、生体分子の反応性基Zとの間の最終的な共有結合形成反応を含む。この反応は、式(V)のタンパク質−生体分子コンジュゲート組成物の形成をもたらす。上述するように、反応性基BおよびZの選択は、ステップ(c)に適した反応条件に影響する。NCL反応条件およびSpyTag−SpyCatcher反応条件はともに、当該技術分野で周知であり、本開示のステップ(c)反応において有用である。(例えば、Dawsonら、(1994)Science 266、776〜779、ZakeriおよびHowarth(2010)JACS 132:4526〜7、およびLiら(2014)J.Mol.Biol.23;426(2):309〜317を参照のこと)。
クリック化学およびネイティブケミカルライゲーションを使用した細孔形成性タンパク質の、ポリメラーゼへの部位選択的コンジュゲーション
[0119]この実施例は、本明細書において開示するステップ(a)〜(c)の部位選択的コンジュゲーション方法の使用について説明し、ここで、BおよびZ反応性基は、ステップ(c)においてネイティブケミカルライゲーション(NCL)を起こす。この実施例は、図1において模式的に描写するように、七量体ナノポア複合体の一部であるα−HL−C46細孔形成性タンパク質のシステイン側鎖がDNAポリメラーゼ(Pol)のN末端にコンジュゲートされる式(V)の組成物の調製を実証する。
[0121]A.細孔形成性タンパク質(例えば、α−HL)精製:使用する細孔形成性タンパク質モノマーは、ともに精製用の6−Hisタグとともにコードされる天然α−HLおよび操作されたα−HL−C46である。6−Hisタグを有する黄色ブドウ球菌(S.aureus)α−HLモノマーのK46C(システインで置換された46位のリジン)変異体(「α−HL−C46」)は、標準的なタンパク質工学技術を使用して調製される(例えば、Valevaら(2001)およびPalmerら(1993)を参照のこと)。天然α−HLおよびα−HL−C46モノマーは、大腸菌(E.coli)において組換え的に発現されて、標準的な技術を使用して、アフィニティー精製される。簡潔に述べると、野生型α−HLおよびα−HL−C46は、「PrepEase」Hisタグ付きタンパク質精製キット(USB−Affymetrix、USA)に関するプロトコールに記載されるように精製し、1.0mg/mLのタンパク質濃度で、pH7.2の1mMトリス−カルボキシエチル−ホスフィン(TCEP)を有する1×PBSに交換する。α−HL精製ステップは全て、還元剤(TCEPまたはDTT)の存在下で実施される。
クリック化学およびSpyCatcher−SpyTag反応を使用した細孔形成性タンパク質の、ポリメラーゼへの部位選択的コンジュゲーション
[0128]この実施例は、ステップ(c)においてSpyTagペプチドの、SpyCatcherタンパク質への反応を提供するBおよびZ反応性基を用いた本明細書において開示するステップ(a)〜(c)の部位選択的コンジュゲーション方法の使用について説明する。この実施例は、図2において模式的に示すように、七量体ナノポア複合体の一部であるα−HL−C46細孔形成性タンパク質のSpyTag修飾C46残基がSpyCatcher−Pol6 DNAポリメラーゼ融合物に部位特異的にコンジュゲートされる式(V)の組成物の調製を実証する。
[0130]A.細孔形成性タンパク質(例えば、α−HL)精製:使用する細孔形成性タンパク質モノマーは、ともに精製用の6−Hisタグとともにコードされる天然α−HLおよび操作されたα−HL−C46である。6−Hisタグを有する黄色ブドウ球菌(S.aureus)α−HLモノマーのK46C(システインで置換された46位のリジン)変異体(「α−HL−C46」)は、標準的なタンパク質工学技術を使用して調製される(例えば、Valevaら(2001)およびPalmerら(1993)を参照のこと)。天然α−HLおよびα−HL−C46モノマーは、大腸菌(E.coli)において組換え的に発現されて、標準的な技術を使用して、アフィニティー精製される。簡潔に述べると、野生型α−HLおよびα−HL−C46は、「PrepEase」Hisタグ付きタンパク質精製キット(USB−Affymetrix、USA)に関するプロトコールに記載されるように精製し、1.0mg/mLのタンパク質濃度で、pH7.2の1mMトリス−カルボキシエチル−ホスフィン(TCEP)を有する1×PBSに交換する。α−HL精製ステップは全て、還元剤(TCEPまたはDTT)の存在下で実施される。
ナノポアアレイにおける実施例2で調製するようなα−HL−Pol6 SpyTag−SpyCatcherコンジュゲートを使用したナノポアシーケンシング
[0137]この実施例は、核酸をシーケンシングするためのナノポアアレイにおける、実施例2で見られるように調製されるα−HL−Pol6ナノポアコンジュゲートの使用について説明する。α−HL−Pol6ナノポアコンジュゲートは、個々にアドレス可能な集積回路チップのアレイ上に形成される膜中に埋め込まれている。このα−HL−Pol6ナノポアアレイは、JAM1A自己プライミングDNA鋳型および4つのヌクレオチドdA、dC、dGおよびdTに相当する4つの異なる5’にタグ付けされたヌクレオチド基質の組に曝露される。DNA鋳型に相補的である特異的な5’−タグ付きヌクレオチドは、Pol6ポリメラーゼ活性部位に捕捉および結合されるため、タグ部分の「尾部」は、すぐ近くにコンジュゲートされるα−HLナノポアの中に配置されるようになる。AC電位の印加の下で、細孔におけるタグの存在は、開放細孔電流(即ち、ナノポアにおいてタグを有さない場合の電流)と比較して特有の阻止電流を引き起こす。Pol6が鋳型に相補的な鎖を合成する場合に測定される阻止電流の配列により、DNA鋳型の配列が同定される。
Claims (16)
- タンパク質および生体分子のコンジュゲートを調製する方法であって、
(a)適切な反応条件下で、チオール基を含むタンパク質を、式(I)
Aは、チオール反応性基であり、
LAは、リンカーであり、
Xは、クリック化学反応性基である)
の化合物と接触させ、それによって、式(II)
の修飾タンパク質を形成するステップ、
(b)式(II)の修飾タンパク質を、式(III)
Bは、反応性基であり、
LBは、リンカーであり、
Yは、式(II)の化合物の同族クリック化学反応性基Xとクリック化学反応を起こすクリック化学反応性基である)
の化合物と接触させ、それによって、構造式(IV)
(c)式(IV)の修飾タンパク質を、適切な条件下で、反応性基Bと共有結合を形成することが可能である反応性基Zを含む生体分子と接触させ、それによって、式(V)
を含み、
前記反応性基Bが、SpyTagペプチドを含むものであり、前記反応性基Zが、SpyCatcherタンパク質を含むものである、前記方法。 - チオール反応性基Aが、マレイミドまたはハロアセトアミドである、請求項1に記載の方法。
- 前記SpyTagペプチドおよび前記SpyCatcherタンパク質が各々、化膿性連鎖球菌(Streptococcus pyogenes)フィブロネクチン結合タンパク質FbaB由来のCnaB2ドメインのアミノ酸配列の断片を含む、請求項1または2に記載の方法。
- チオール反応性基Aが、マレイミドまたはハロアセトアミドであり、ハロゲン原子が、F、Cl、Br、およびIから選択される、請求項4に記載の組成物。
- クリック化学反応性基XおよびYが、クリック化学反応性基の下記対:アジドおよびアルキン、アジドおよびシクロオクチン、ならびにアジドおよびジベンゾシクロオクチン−アミンから選択される対である、請求項4または5に記載の組成物。
- 前記SpyTagペプチドが、化膿性連鎖球菌(Streptococcus pyogenes)フィブロネクチン結合タンパク質FbaB由来のCnaB2ドメインのアミノ酸配列の断片を含む、請求項4〜7のいずれか一項に記載の組成物。
- チオール反応性基Aが、マレイミドである、請求項9に記載の組成物。
- クリック化学反応性基XおよびYが、クリック化学反応性基の下記対:アジドおよびアルキン、アジドおよびシクロオクチン、ならびにアジドおよびジベンゾシクロオクチン−アミンから選択される対である、請求項9または10に記載の組成物。
- SpyTagペプチドおよびSpyCatcherタンパク質が各々、化膿性連鎖球菌(Streptococcus pyogenes)フィブロネクチン結合タンパク質FbaB由来のCnaB2ドメインのアミノ酸配列の断片を含む、請求項9〜12のいずれか一項に記載の組成物。
- タンパク質が、任意選択で、α−溶血素、β−溶血素、γ−溶血素、アエロリジン、溶血毒、ロイコシジン、メリチン、MspAポリンおよびポリンAからなる群から選択される細孔形成性タンパク質である、請求項9〜13のいずれか一項に記載の組成物。
- タンパク質が、膜中に埋め込まれた細孔形成性タンパク質である、請求項9〜14のいずれか一項に記載の組成物。
- 生体分子が、任意選択で、DNAポリメラーゼ、RNAポリメラーゼ、逆転写酵素、およびDNAリガーゼからなる群から選択される、ポリマーの合成を触媒することが可能な酵素である、請求項9〜15のいずれか一項に記載の組成物。
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