JP6867302B2 - タンパク質の特徴を改善する方法 - Google Patents
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- JP6867302B2 JP6867302B2 JP2017556995A JP2017556995A JP6867302B2 JP 6867302 B2 JP6867302 B2 JP 6867302B2 JP 2017556995 A JP2017556995 A JP 2017556995A JP 2017556995 A JP2017556995 A JP 2017556995A JP 6867302 B2 JP6867302 B2 JP 6867302B2
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- C12N15/09—Recombinant DNA-technology
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Description
こうしたことから、複雑性が抑えられた代表的なライブラリーを構築するための効率的な技術が利用可能となれば、そのような技術によって候補結合化合物の特性を迅速に評価/選択することが可能となり、タンパク質結合反応の理解を必要とする努力、例えばタンパク質や抗体のエンジニアリング等が進歩するであろう。
本発明の態様および実施形態を、いくつかの実施事例および適用において例示し、そのいくつかを以下に、本明細書全体を通じて要約する。
本発明の実践法は、別途明示しない限り、従来技術および当技術分野の技能に属する有機化学、分子生物学(組換え技術を含む)、細胞生物学、および生化学の記述を採用し得る。そのような従来技術として、合成ポリヌクレオチドの調製、モノクロナール抗体、抗体ディスプレイシステム、核酸配列決定法、および分析法等が挙げられるが、但しこれらに限定されない。適する技術の具体的な説明は、本明細書の下記の実施例を参照することにより得られるが、但しその他の同等の従来手順も、もちろん利用可能である。そのような従来技術および説明は、標準的な研究室マニュアル、例えばGenome Analysis: A Laboratory Manual Series (I〜IV巻);PCR Primer: A Laboratory Manual;Phage Display: A Laboratory Manual;およびMolecular Cloning: A Laboratory Manual(すべてCold Spring Harbor Laboratory Press刊);Sidhu編、Phage Display in Biotechnology and Drug Discovery (CRC Press、2005年);LutzおよびBornscheuer編、Protein Engineering Handbook (Wiley-VCH、2009年);Hermanson、Bioconjugate Techniques、第2版(Academic Press、2008年)等に見出され得る。
参照タンパク質(配列番号1):
… -Gln-Ala-Ala-Phe-[Glu-Lys-Thr]-Ser-Ala-His-Lys-Met- …
参照タンパク質の核酸配列(配列番号2):
… -CAA-GCA-GCA-TTC-[GAG-AAA-ACG]-TCA-GCC-CAC-AAG-ATG- …
12−ヌクレオチドの重複を有するGlu−Lys−Thrドメインに関する単一置換ライブラリーのメンバー(配列番号3、配列番号4、配列番号5):
CAA-GCA-GCA-TTC-[NNN-AAA-ACG]-TCA-GCC-CAC-AAG
CAA-GCA-GCA-TTC-[GAG-NNN-ACG]-TCA-GCC-CAC-AAG
CAA-GCA-GCA-TTC-[GAG-AAA-NNN]-TCA-GCC-CAC-AAG
この場合、「NNN」は、以下に記載するように「ワイルドカード」コドンである。
(物理的、化学的、および生物学的特徴を改善するための選択)
(タンパク質ディスプレイシステム)
(核酸によりコードされる結合化合物のライブラリー)
43個のファージミドライブラリーインサートを、図2Aに示すsdFvをコードするDNA配列を含め合成した。各インサートは、アミノ酸(下線部)の1つについて、縮重コドンNNNと置き換わったコドンを有した。これらのインサートをファージミドにクローン化して、それぞれ19個の変異体sdFvおよび1個の野生型をコードする43個のミニライブラリーを生成した。これらのミニライブラリーをE.coli系統SS320に形質転換し、そして形質転換体を混合して、sdFvのCDR1、CDR2、およびCDR3を含む3つのサブライブラリーとした。これらのサブライブラリーを増殖させ、そしてM13K07ヘルパーファージで感染し、そして3つのファージライブラリーを生成した。
(定義)
Claims (6)
- タンパク質のタンパク質結合部位を改善する方法であって、
タンパク質結合部位の複数のドメインのそれぞれについて、個別の単一置換ライブラリーを合成して、複数の個別の単一置換ライブラリーを与えるステップであって、単一置換ライブラリーの各メンバーが、異なる単一置換ライブラリーの少なくとも1つのメンバーのヌクレオチド配列と重複するヌクレオチド配列を有し、前記タンパク質結合部位の複数のドメインが1〜250アミノ酸位置で個々に置換される、ステップと、
各単一置換ライブラリーの各メンバーを、事前候補タンパク質として個別に発現させるステップと、
標的分子に結合する事前候補タンパク質をコードする、各単一置換ライブラリーのメンバーを選択して、前記タンパク質のドメイン毎に選択されたライブラリーを形成するステップと、
PCRにおいて、前記選択されたライブラリーのメンバーをシャッフリングして、コンビナトリアルシャッフルドライブラリーを生成するステップと、
前記シャッフルドライブラリーのメンバーを、候補タンパク質として発現させるステップと、
結合条件下の反応混合物中で前記候補タンパク質を標的分子と一緒にインキュベートするステップと、
候補タンパク質の画分が結合したまま残留するまで前記標的分子を洗浄するステップと、
前記標的分子に結合したまま残留する候補タンパク質をコードする、前記シャッフルドライブラリーのメンバーを選択するステップと
を含む方法。 - 前記結合部位が、タンパク質ディスプレイシステムにより発現される抗体または抗体断片の結合部位である、請求項1に記載の方法。
- 前記タンパク質ディスプレイシステムが、酵母菌ディスプレイシステム、哺乳動物ディスプレイシステム、細菌ディスプレイシステム、昆虫細胞ディスプレイシステム、またはファージディスプレイシステムである、請求項2に記載の方法。
- 前記タンパク質ディスプレイシステムが、ファージディスプレイシステムである、請求項3に記載の方法。
- 前記複数のドメインが、前記タンパク質の連続したアミノ酸配列をカバーする、請求項1に記載の方法。
- 前記結合したまま残留する候補タンパク質の画分が10%である、請求項1〜5のいずれか一項に記載の方法。
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