JP6902052B2 - 複数のリガーゼ組成物、システム、および方法 - Google Patents
複数のリガーゼ組成物、システム、および方法 Download PDFInfo
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- JP6902052B2 JP6902052B2 JP2018560726A JP2018560726A JP6902052B2 JP 6902052 B2 JP6902052 B2 JP 6902052B2 JP 2018560726 A JP2018560726 A JP 2018560726A JP 2018560726 A JP2018560726 A JP 2018560726A JP 6902052 B2 JP6902052 B2 JP 6902052B2
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- ligase
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Description
本願明細書は、複数のリガーゼを使用する組成物、システム、および方法であって、リガーゼの少なくとも1つはアデニル化欠損ATP依存性リガーゼまたは非アデニル化ATP依存性リガーゼ(例えば、ATP非含有混合物中に存在する)である、組成物、システム、および方法を提供する。1つの態様において、複数のリガーゼは、プレアデニル化二本鎖配列を非アデニル化二本鎖配列にライゲートする(例えば、アデニル化欠損ATP依存性リガーゼまたは非アデニル化ATP依存性リガーゼは第1の鎖にライゲートし、異なるリガーゼは第2の鎖にライゲートする)ために使用される。他の態様において、本願明細書において変異体T4リガーゼ(例えば、K159S変異体またはK159C変異体)が提供される。
DNAリガーゼは、ATPまたはNAD+を利用し、3’−ヒドロキシルおよび5’−ホスフェートを有する隣接ポリヌクレオチド末端間でホスホジエステル結合形成を触媒する二価金属イオン依存性酵素である(Tomkinson AE, PNAS, 2006)。DNAリガーゼは、DNA複製および修復のための重要な酵素であり、真核生物、細菌、古細菌および多数のウイルスにおいて普遍的に見いだされる。種の起源に依存して、天然に存在するDNAリガーゼは、多数のユニークな特性、例えば、基質特異性、配列およびドメイン構成、最適な反応条件、例えばpH、温度および耐塩性を有し得る。
本願明細書は、複数の異なるリガーゼを使用する組成物、システム、キット、および方法であって、リガーゼの少なくとも1つはアデニル化欠損ATP依存性リガーゼまたは非アデニル化ATP依存性リガーゼ(例えば、ATP非含有混合物中に存在する)である、組成物、システム、キット、および方法を提供する。1つの態様において、複数のリガーゼは、プレアデニル化二本鎖配列を非アデニル化二本鎖配列にライゲートする(例えば、アデニル化欠損ATP依存性リガーゼまたは非アデニル化ATP依存性リガーゼは第1の鎖にライゲートし、異なるリガーゼは第2の鎖にライゲートする)ために使用される。他の態様において、本願明細書において変異体T4リガーゼ(例えば、K159S変異体またはK159C変異体)が提供される。
本願明細書において使用される「アデニル化欠損ATP依存性リガーゼ」なる用語は、ATP依存性リガーゼによって通常行われるとおりATPと反応することによってAMP−リガーゼ中間体を形成することができないATP依存性リガーゼを示す。かかるDNAリガーゼの例は、T4 DNAリガーゼ K159S、K159C、およびK159A変異体、ならびにChlorella ウイルス PBCV−1 DNAリガーゼからのK27A変異体を含むが、これらに限定されない。例はまた、リガーゼの他の保存されたモチーフにおける突然変異誘発、例えば、T4 RNA リガーゼ2におけるR55K、K227QおよびK225Rを含む(Yin et al., J Biol Chem 2003, 278:17601-17608、出典明示によりその全体において、特にかかる変異体について本願明細書に包含させる)。アデニル化欠損ATP依存性リガーゼの他の例は、リガーゼのKxDG(モチーフI)内のリジンが、リガーゼがアデニル化欠損であるが、ステップ3リガーゼが有能であるように他の19アミノ酸の1つに変異されるATP依存性リガーゼを含む。かかる変異体は、産生され、例えば以下の実施例の部において、示される方法を使用して、アデニル化欠損およびステップ3リガーゼ有能についてスクリーニングすることができる。
本願明細書は、複数のリガーゼを使用する組成物、システム、および方法であって、リガーゼの少なくとも1つはアデニル化欠損ATP依存性リガーゼまたは非アデニル化ATP依存性リガーゼ(例えば、ATP非含有混合物中に存在する)である、組成物、システム、および方法を提供する。1つの態様において、複数のリガーゼは、プレアデニル化二本鎖配列を非アデニル化二本鎖配列にライゲートする(例えば、アデニル化欠損ATP依存性リガーゼまたは非アデニル化ATP依存性リガーゼは第1の鎖にライゲートし、異なるリガーゼは第2の鎖にライゲートする)ために使用される。他の態様において、本願明細書において変異体T4リガーゼ(例えば、K159S変異体またはK159C変異体)が提供される。
活性なアデニル化欠損ステップ−3−卓越T4 DNAリガーゼ変異体の同定
野生型T4 DNAリガーゼ(T4DL)をN−末端6xHisタグを有する最適化大腸菌コドンを使用して合成した。合成遺伝子をT7−プロモーター駆動発現ベクターpTXB1(NEB)にクローニングした。鋳型としてpTXB1_T4DLWTを使用するPCRベースの突然変異誘発を行い、リジン159を他の全ての19個のアミノ酸に変化させた。全ての変異体発現ベクターを配列確認し、予期される変化を確認した。次に、各変異体をT7 Express株(#C2566、NEB)において発現させた。簡潔には、各6ml培養物を最初にO.D.〜0.6に増殖させ、IPTGを0.5mMの最終濃度に誘導させ、20℃で一晩振盪震盪した。次に、発現培養物を遠沈し、20mM Tris(pH=7.5)、150mM NaCl、1x FastBreak(Promega)、200ug/ml リゾチームを含む450ul バッファー中に再懸濁し、超音波処理した。次に、細胞溶解物を4℃で30分間14,000rpmで遠心した。清澄化された溶解物をNi−NTAビーズカラム上に負荷し、標的タンパク質を20mM Tris(pH=7.5)、150mM NaCl、400mM イミダゾールを含む溶出バッファーを使用して溶出させた。
Claims (3)
- T4 DNAリガーゼ K159S変異体を含む組成物であって、アミノ酸159は配列番号:1の位置159でのセリンであり、変異体は配列番号:1のアミノ酸配列と少なくとも90%同一性を有し、リガーゼ活性を有する、組成物。
- K159S変異体が配列番号:1のアミノ酸配列からなる、請求項1に記載の組成物。
- a)T4 DNAリガーゼ K159S変異体を含む第1の組成物を含む第1の容器、ここでアミノ酸159は配列番号:1の位置159でのセリンであり、変異体は配列番号:1のアミノ酸配列と少なくとも90%同一性を有し、リガーゼ活性を有する;および
b)アデニル化二本鎖核酸配列(ADSNAS)を有する核酸を含む第2の組成物を含む第2の容器
を含むキット。
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