JP2008508871A - ポリペプチドのc末端修飾 - Google Patents
ポリペプチドのc末端修飾 Download PDFInfo
- Publication number
- JP2008508871A JP2008508871A JP2007524304A JP2007524304A JP2008508871A JP 2008508871 A JP2008508871 A JP 2008508871A JP 2007524304 A JP2007524304 A JP 2007524304A JP 2007524304 A JP2007524304 A JP 2007524304A JP 2008508871 A JP2008508871 A JP 2008508871A
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- JP
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- Prior art keywords
- peptide
- xaa
- trypsin
- amino acid
- mutant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Abstract
Description
Pn _P3 _P2 _P1 _P1`_P2`_P3`_Pn`
Sn _S3 _S2 _S1 _S1`_S2`_S3`_Sn`
トリプシンバリアント作製の一般的手順:
1. トリプシンまたはトリプシノーゲンをコードするDNAを含む適当なベクター、例えばpSTベクター(図1参照)を用いるトリプシンまたはトリプシノーゲン内への所望の突然変異の導入。大腸菌ベクターpSTは、もともと、L. Hedstromから構築され、ADH/GAPDH-プロモーターおよびトリプシノーゲンをコードする配列と融合させたα-因子リーダー配列を含有する酵母シャトルベクターを表す; Hedstrom, Lら, Science 255 (1992) 1249-1253を参照のこと。
2. 構築されたベクターの、例えば大腸菌内での形質転換。
3. それぞれ適当な発現ベクター、例えば酵母ベクターpYT (図1参照)を用いた、修飾トリプシン配列およびトリプシノーゲン配列のサブクローニング。所望の突然変異をこの発現ベクターに直接導入した場合、この工程は必要でない。
4. 大腸菌または酵母での修飾トリプシンまたはトリプシノーゲンの発現。
5. 適当な分離法、例えばカチオン交換クロマトグラフィーを用いる、修飾トリプシンまたはトリプシノーゲンの単離。
6. トリプシノーゲンが発現された場合は、単離されたチモーゲンは、エンテロキナーゼによる限定的なタンパク質分解によって活性化する必要がある。
7. 適当な精製法、例えばアフィニティクロマトグラフィーまたはアニオン交換クロマトグラフィーを適用する活性化されたトリプシンの最終精製。
8. 透析
[理論的pI: 5.41/Mw (平均質量): 23843.00/Mw (モノアイソトピック質量): 23827.59]
固相ペプチド合成によるペプチドの調製
別途指定する場合を除き、本出願において記載するペプチドは、バッチペプチド合成装置、例えばApplied BiosystemsのA433で、フルオレニルメチルオキシカルボニル-(Fmoc)-固相ペプチド合成により合成した。この方法では、各場合において、表2に示すアミノ酸誘導体4.0当量を使用した。
トリプシンバリアント Tn K60E、E151H、N143H、D189Kによって触媒される、Arg-NH2によるAla-Ala-Tyr-Arg-His-Ala-Glyのアミノ基転移
ペプチドAla-Ala-Tyr-Arg-His-Ala-Glyを、実施例2に記載のようにFmoc-化学を用いた従来の固相ペプチド合成および予め加えたWang-樹脂によって合成した。それぞれのアミノ酸構成ブロックは市販されており、種々の供給業者から購入した。Arg-NH2は、Bachem (スイス)の市販品であった。
トリプシンバリアントTn K60E、E151H、N143H、D189Kの特異性に対するZn2+イオンの影響
ペプチド基質Bz-Ala-Ala-Tyr-Arg-His-Ala-Ala-Gly、Bz-Ala-Ala-Tyr-Arg-His-Ala-Gly、Bz-Ala-Ala-Tyr-Arg-His-Asp-Ala-Gly、Bz-Ala-Ala-Tyr-Arg-Arg-Ala-Gly、およびBz-Ala-Ala-Tyr-Asp-His-Ala-Glyを、Fmoc-化学を用いた従来の固相ペプチド合成および予め加えたWang-樹脂によって合成した。それぞれのアミノ酸構成ブロックは市販されており、種々の供給業者から購入した。
トリプシンバリアント Tn K60E、E151H、N143H、D189Kにより触媒される反応速度に対する認識配列の影響
配列番号: 3〜19のNα-ベンゾイル化ペプチドを、Fmoc-化学を用いた従来の固相ペプチド合成および予め加えたWang-樹脂により合成した。それぞれのアミノ酸構成ブロックは市販されており、種々の供給業者から購入した。
トリプシンバリアント Tn K60E、E151H、N143H、D189Kによって触媒されるArg-His-Ala-Lys(6-CF)-OHによるBz-Ala-Ala-Tyr-Arg-His-Ala-Glyのアミノ基転移
Bz-Ala-Ala-Tyr-Arg-His-Ala-Glyは、Fmoc-化学を用いた従来の固相ペプチド合成および予め加えたWang-樹脂により合成した。それぞれのアミノ酸構成ブロックは市販されており、種々の供給業者から購入した。Arg-His-Ala-Lys(6-CF)-OH を、フラグメント縮合(fragment condensation)によって合成した。保護されたトリペプチドBoc-Arg(Boc)2-His(Trt)-Ala-OHを、従来の固相ペプチド合成を用いてクロロトリチル-樹脂上で合成した。塩化メチレン/酢酸/トリフルオロ酢酸 (v/v/v 8/1/1)を含む40 mlのカクテルで2回、ペプチドを樹脂から切断した。粗製物を、逆相HPLCにより精製した。他のフラグメントFmoc-Lys(6-カルボキシ-フルオレセイン)の合成を、3 mlのジオキシンおよび3 mlのDMF中、0.6 mmol Fmoc-Lys*HCl、0,655 mMol 6-カルボキシフルオレセイン (Molecular Probesから購入)から行なった。ピペリジンおよび逆相HPLCにより精製した粗製物を用いてFmoc-基を切断した。次いで、保護されたトリペプチドを、DMF中1当量のHBTU (Iris Biotech)および3当量のジイソプロピルエチルアミンで活性化した。1当量のLys(6-カルボキシ-フルオレセイン)を添加し、混合物を室温で2時間攪拌した。次いで、脱保護カクテル(18 mlのトリフルオロ酢酸、0.5 mlの水および0.5 mlのエタンジチオール)を用いて脱保護を行なった。ペプチドを、ジイソプロピルエーテルを用いて沈殿させ、RP-HPLCにより精製し、良好な収率で得た。
トリプシンバリアント Tn K60E、E151H、N143H、D189Kによって触媒されるArg-His-Gly-PEGによるAKTAAALHIL VKEEKLALDL LEQIKNGADF GKLAKKHSIC PSGKRGGDLG EFRQGQMVPA FDKVVFSCPV LEPTGPLHTQ FGYHIIKVLY RHのアミノ基転移
Arg-His-Gly-PEGを、Boc-Arg(Boc)2-His(Trt)-Gly-OHのフラグメント縮合およびNektar/Shearwaterから購入したアミノ-PEG (20kD)により合成した。保護されたトリペプチドの合成およびフラグメントの活性化については実施例6を参照のこと。ここでは、0.5当量のアミノ-PEG (20 kD)を、求核試薬として用いた。脱保護後(カクテルについては実施例6を参照)、RP-HPLCを用いてすべての低分子不純物を分離した。
Claims (9)
- K60位およびD189位の両方にアミノ酸置換、ならびにN143位またはE151位にヒスチジンによる少なくとももう1つ以上のアミノ酸置換を含む突然変異トリプシン。
- K60がEまたはDで置換されている、請求項1記載の突然変異トリプシン。
- D189がK、HまたはRで置換されている、請求項1記載の突然変異トリプシン。
- 標的ペプチド、および切断部位Xaa1-Xaa2-His(式中、Xaa1はL、YまたはFであり、Xaa2はRまたはKである)を含む制限部位ペプチドを含み、前記制限部位ペプチドが前記標的ペプチドのC末端のアミノ酸Xaa1で標的ペプチドと重複する、請求項1〜3いずれか記載のトリプシン突然変異体の基質としてのポリペプチドの使用。
- (a) 標的ペプチド、および切断部位Xaa1 _Xaa2 _His(式中、Xaa1はL、Y、またはFであり、Xaa2はRまたはKである)を含む制限部位ペプチドを含み、前記制限部位ペプチドが前記標的ペプチドのC末端のアミノ酸Xaa1で標的ペプチドと重複するポリペプチドを提供する工程、
(b) 前記ペプチドを、Xaa1後の内部タンパク質分解性切断およびエンドプロテアーゼ標的ペプチドペプチド-アシル-中間体の形成を可能にする条件下で、請求項1〜3いずれか記載のトリプシン突然変異体と接触させる工程、
(c) 適切な求核試薬を添加する工程、および
(d) 求核性攻撃および標的ペプチドのC末端への前記求核試薬の結合時、エンドプロテアーゼ標的ペプチドアシル-中間体から突然変異トリプシンを放出する工程
を含む、C末端がアシル基転移した標的ペプチドの作製方法。 - 前記求核試薬が、第1級アミン、イミン、第2級アミン、チオールおよびヒドロキシル基からなる群より選択される、請求項5記載の方法。
- 修飾を含む前記求核試薬が、アミノ酸アミド、ペプチド、ペプチドアミド、標識、標識されたアミノ酸アミド、標識されたペプチド、標識されたペプチドアミド、およびポリエチレングリコールからなる群より選択される、請求項5記載の方法。
- 前記修飾がポリエチレングリコールである、請求項7記載の方法。
- 請求項1〜3いずれか記載の突然変異トリプシンをコードするヌクレオチド配列。
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JP2013518922A (ja) * | 2010-02-11 | 2013-05-23 | エフ.ホフマン−ラ ロシュ アーゲー | Igf−iポリ(エチレングリコール)コンジュゲート |
JP2022514951A (ja) * | 2018-12-19 | 2022-02-16 | バイオファーマ トランスレーションズインスティテュート デッサウ フォルシュングス ゲーエムベーハー | 酵素特性が改善したトリプシン変異体 |
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JP2013518922A (ja) * | 2010-02-11 | 2013-05-23 | エフ.ホフマン−ラ ロシュ アーゲー | Igf−iポリ(エチレングリコール)コンジュゲート |
JP2015145402A (ja) * | 2010-02-11 | 2015-08-13 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Igf−iポリ(エチレングリコール)コンジュゲート |
JP2022514951A (ja) * | 2018-12-19 | 2022-02-16 | バイオファーマ トランスレーションズインスティテュート デッサウ フォルシュングス ゲーエムベーハー | 酵素特性が改善したトリプシン変異体 |
JP7360742B2 (ja) | 2018-12-19 | 2023-10-13 | バイオファーマ トランスレーションズインスティテュート デッサウ フォルシュングス ゲーエムベーハー | 酵素特性が改善したトリプシン変異体およびその使用ならびに基質の直交二重修飾のための方法 |
US12030925B2 (en) | 2019-05-17 | 2024-07-09 | Bioverativ Therapeutics Inc. | Methods of treating hemophilia A |
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CA2569707C (en) | 2013-09-24 |
US8927230B2 (en) | 2015-01-06 |
WO2006015879A1 (en) | 2006-02-16 |
JP4538501B2 (ja) | 2010-09-08 |
ATE400647T1 (de) | 2008-07-15 |
CA2569707A1 (en) | 2006-02-16 |
EP1778839A1 (en) | 2007-05-02 |
EP1778839B1 (en) | 2008-07-09 |
US20080064079A1 (en) | 2008-03-13 |
CN101010425B (zh) | 2010-05-26 |
CN101010425A (zh) | 2007-08-01 |
ES2309785T3 (es) | 2008-12-16 |
DE602005008071D1 (de) | 2008-08-21 |
US20130177940A1 (en) | 2013-07-11 |
HK1110888A1 (en) | 2008-07-25 |
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