JP4303283B2 - コンジュゲート用の脱グリコシル化された酵素 - Google Patents
コンジュゲート用の脱グリコシル化された酵素 Download PDFInfo
- Publication number
- JP4303283B2 JP4303283B2 JP2006500062A JP2006500062A JP4303283B2 JP 4303283 B2 JP4303283 B2 JP 4303283B2 JP 2006500062 A JP2006500062 A JP 2006500062A JP 2006500062 A JP2006500062 A JP 2006500062A JP 4303283 B2 JP4303283 B2 JP 4303283B2
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- Japan
- Prior art keywords
- enzyme
- alkaline phosphatase
- binding
- conjugate
- target molecule
- 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.)
- Expired - Lifetime
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Description
組み換えによって産生されたアルカリホスファターゼの脱グリコシル化
ピキア・パストリスから単離した10mgの組み換えによって産生されたアルカリホスファターゼ(図中では「recAP」ともいわれる)を、200ミリ単位のエンドグリコシダーゼH(「endoH」、Rocheカタログ番号1643053)と共に、1mMのMgCl2と1.5MのNaClを含む1mlの30mM酢酸ナトリウム緩衝液(pH5.5)中で2時間インキュベートした。この時間の後、溶液を、YM30膜を取り付けた濾過チャンバー中で、150mMのNaCl、1mMのMgCl2、および0.1mMのZnCl2を含むトリエタノールアミン/HCl緩衝液(pH7.6)を使用して徹底的に透析し、これによって1mlの最終容量を得た。図1は、endo Hでの処理の前および後の、組み換えによって産生されたアルカリホスファターゼのTSK 3000カラム上での溶出時間を示す。処理前の6.88分での溶出ピークが完全に消滅しており、新しい溶出ピークが7.61分に現れたことを見ることができる。このことは、組み換えによって産生されたアルカリホスファターゼがグリカン鎖を失い、その結果、変化していない分子と比較して、より分子量が小さくなり、溶出時間が遅くなったことを示している。
AP酵素活性の測定
アルカリホスファターゼの酵素活性を、記載されている試験プロトコール(Z. Klin. Chem. Klin. Biochem. 8(1970)658-660;およびZ. Klin. Chem. Klin. Biochem. 10(1972)182)にしたがって基質として4−ニトロフェニルホスフェートを使用して決定した。組み換えによって産生されたピキア・パストリス由来アルカリホスファターゼは、約7,000U/mgの比活性を示し、これはエンドグリコシダーゼHでの処理によって変化しなかった。
アルカリホスファターゼコンジュゲートの調製
APの活性化
0.5mlの30mMリン酸ナトリウム緩衝液(pH7.1)中に溶解させたアルカリホスファターゼ調製物(組み換えによって産生されたアルカリホスファターゼ;組み換えによって産生されたアルカリホスファターゼ/endo Hで処理したもの)各5mgを、12μlのDMSO中に溶解させた0.059mgのN−スクシンイミジル−S−アセチルチオプロピオナート(SATP)と混合し、室温(RT)で1時間攪拌した。反応を、5μlの1Mリジン−HClの添加によって停止させ、室温(RT)でさらに30分間攪拌した。その後、反応混合物を、50mMのNaClを含む1.5lの10mMリン酸カリウム緩衝液(pH6.1)に対して、4℃で一晩、徹底的に透析した。
1mlの30mMリン酸ナトリウム緩衝液(pH7.1)中に溶解させた20mgのモノクローナル抗体である抗hCG−M−INN22−IgGを、21μlのDMSO中に溶解させた0.205mgのマレイミドヘキサノイル−N−ヒドロキシスクシンイミドエステル(MHS)と混合し、RTで1時間攪拌した。反応を、5μlの1Mリジン−HClの添加によって停止させ、RTでさらに30分間攪拌した。その後、反応混合物を、50mMのNaClを含む5lの10mMリン酸カリウム緩衝液(pH6.1)に対して、4℃で一晩、徹底的に透析した。
50mMのNaClを含む324μlの10mMリン酸カリウム緩衝液(pH7.5)中に溶解させた、SATPで活性化したAP調製物(組み換えによって産生されたアルカリホスファターゼ;組み換えによって産生されたアルカリホスファターゼ/endo Hで処理したもの)各3mgを、8μlの1Mヒドロキシルアミン溶液と混合し、RTで1時間攪拌した。この時間の後、50mMのNaClを含む262μlの10mMリン酸カリウム緩衝液(pH7.5)中に溶解させた1.74mgのMHS活性化抗hCG−M−INN22−IgGを添加し、反応混合物をRTで2時間攪拌した。その後、7μlの0.2Mシステイン−HCl溶液(1Mのリン酸の添加によってpH6.7に調整した)を添加し、RTで30分間攪拌した。この後、7μlの0.5M N−メチルマレイミド水溶液を添加し、RTでさらに30分間攪拌した。次いで、反応混合物を、150mMのNaCl、1mMのMgCl2、および0.1mMのZnCl2を含む1.5lの50mMのトリエタノールアミン−HCl緩衝液(pH7.6)に対して、4℃で一晩、徹底的に透析した。透析後、溶液を3MのNaCl濃度とし、使用するまで+4℃で保存した。
hCG ELISAによるコンジュゲートの比較
hCG ELISAを、ストレプトアビジンでコーティングしたマイクロタイタープレートを使用することによって、サンドイッチアッセイとして行った。ビオチニル化Mab抗hCG−M−1F79−Fab誘導体を、固定化した結合パートナーとして使用した(1ウェルあたり、インキュベーションバッファー中の5μg/ml溶液150μlをRTで30分間インキュベートした)。3回の洗浄後、それぞれ、インキュベーションバッファー中に溶解させた、0、14.84、254.8、2103、および7801mU/mlの濃度の120μlのhCG試料を、それぞれのウェルに添加し、RTで1時間インキュベートした。3回の洗浄後、100μlの上記アルカリホスファターゼ/Mab抗hCG−M−INN22−IgGコンジュゲートをそれぞれ添加し、RTで1時間インキュベートした。3回の洗浄後、1ウェルあたり100μlの100mM 4−ニトロフェニル−ホスフェート基質溶液を添加し、RTで20分間のインキュベーションの後、吸光度を、補正波長として490nmを使用して、405nmでELISAリーダーで測定した。
マトリックス支援レーザー脱離イオン化飛行時間型質量分析装置(MALDI−TOF−MS)によるendo Hで脱グリコシル化したrecAPの分子量の決定
ピキア・パストリス中で産生させたrecAPを、実施例1に記載したようにendo Hによって処理し、蒸留水に対して透析した。タンパク質溶液をシナピン酸マトリックス溶液と混合し、標的上で結晶化させた(chrystallized)。試料を、ディレイドエクストラクションを備えたVoyager Biospectrometryワークステーション上で、ポジティブモードで分析した。
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WO 00/56903
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Claims (13)
- (a)形質転換されたピキア・パストリス酵母における発現により得られ、該酵母から単離した配列番号:1のアミノ酸配列を含むグリコシル化アルカリホスファターゼ酵素を提供する工程、
(b)工程(a)の酵素をエンド−β−N−アセチルグルコサミニダーゼHを使用して部分的に脱グリコシル化する工程、
(c)部分的に脱グリコシル化された酵素を単離する工程、
(d)工程(c)の部分的に脱グリコシル化された酵素を、標的分子に結合可能な分子に結合させる工程
を含む、標的分子に結合可能な分子と酵素とのコンジュゲートの作製方法。 - 工程(b)において、20%〜70%の炭水化物残基がグリコシル化酵素から切断除去されることを特徴とする、請求項1記載の方法。
- 工程(b)において、約60%の炭水化物残基がグリコシル化酵素から切断除去されることを特徴とする、請求項2記載の方法。
- 配列番号:1のアミノ酸配列のグリコシル化部位が、アルカリホスファターゼを発現する形質転換宿主生物中で認識されることを特徴とする、請求項1〜3いずれか記載の方法。
- アルカリホスファターゼが、配列番号:1のアミノ酸配列を含有するアルカリホスファターゼであることを特徴とする、請求項4記載の方法。
- 標的分子に結合可能な分子が、
(a)抗体またはその機能性断片、ここで、該機能性断片は、該機能性断片が由来する全長の抗体の少なくとも1つの結合機能を保持している、
(b)アビジン、またはアビジン分子のポリマー、またはビオチンに結合可能なアビジンの断片、またはビオチンに結合可能なアビジン断片のポリマー、
(c)ストレプトアビジン、またはストレプトアビジン分子のポリマー、またはビオチンに結合可能なストレプトアビジンの断片、またはビオチンに結合可能なストレプトアビジン断片のポリマー、
(d)レクチン、または炭水化物に結合可能なその断片、
(e)ハプテン、
(f)核酸またはそのアナログ、ここで該アナログは、ホスホロチオエートおよびペプチド核酸からなる群より選択される
からなる群より選択されることを特徴とする、請求項1〜5いずれか記載の方法。 - 標的分子に結合可能な分子が、連結基によって酵素に結合することを特徴とする、請求項1〜6いずれか記載の方法。
- (a)形質転換されたピキア・パストリス酵母における発現により得られ、該酵母から単離した配列番号:1のアミノ酸配列を含むグリコシル化アルカリホスファターゼ酵素を提供する工程、ここで、該グリコシル化酵素の炭水化物部分は多数のマンノースサブユニットを含む、
(b)工程(a)の酵素をエンド−β−N−アセチルグルコサミニダーゼHを使用して部分的に脱グリコシル化する工程、
(c)部分的に脱グリコシル化された酵素を単離する工程
を含む方法によって得られ得る、哺乳動物性タンパク質を含まない、哺乳動物起源の、単離および部分的に脱グリコシル化されたアルカリホスファターゼ酵素。 - アルカリホスファターゼのサブユニットの分子量が54kDa〜58kDaであることを特徴とする、請求項8記載の酵素。
- コンジュゲートを形成するための請求項8または9記載の酵素の使用。
- 請求項1〜7いずれか記載の方法により得られ得る、標的分子に結合可能な分子と酵素とのコンジュゲート。
- 標的分子の存在を検出するため、または標的分子の量を測定するためのアッセイにおける請求項11記載のコンジュゲートの使用。
- 固相に結合させた標的分子に結合可能な分子、請求項11記載のコンジュゲート、インキュベーションバッファー、および該コンジュゲートの酵素部分により変換され得る基質を含有してなる、キット。
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EP03005930.7 | 2003-03-17 | ||
EP03005930A EP1460425A1 (en) | 2003-03-17 | 2003-03-17 | Deglycosylated enzymes for conjugates |
PCT/EP2004/002707 WO2004083862A1 (en) | 2003-03-17 | 2004-03-17 | Deglycosylated enzymes for conjugates |
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US (1) | US7482145B2 (ja) |
EP (2) | EP1460425A1 (ja) |
JP (1) | JP4303283B2 (ja) |
AT (1) | ATE368222T1 (ja) |
CA (1) | CA2515686C (ja) |
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EP1460425A1 (en) | 2003-03-17 | 2004-09-22 | Boehringer Mannheim Gmbh | Deglycosylated enzymes for conjugates |
EP2037274A1 (de) * | 2007-09-11 | 2009-03-18 | GALAB Technologies GmbH | Lektinbasierter Glykan-Assay |
CN102695716B (zh) | 2009-07-14 | 2016-03-02 | 独立行政法人产业技术综合研究所 | 肝病病情指标糖链标记物 |
KR101470108B1 (ko) * | 2009-07-14 | 2014-12-05 | 도꾸리쯔교세이호진 상교기쥬쯔 소고겡뀨죠 | 당단백질의 측정 방법, 간질환의 검사 방법, 당단백질 정량용 시약 및 간질환 병태 지표 당쇄 마커 당단백질 |
CN105648502B (zh) * | 2016-03-28 | 2018-04-10 | 桂林理工大学 | 一种镁合金表面疏水复合膜层及其制备方法 |
EP3559249A1 (en) | 2016-12-21 | 2019-10-30 | H. Hoffnabb-La Roche Ag | Method for in vitro glycoengineering of antibodies |
CN110088291A (zh) * | 2016-12-21 | 2019-08-02 | 豪夫迈·罗氏有限公司 | 用于体外糖工程化抗体的方法 |
JPWO2019069977A1 (ja) | 2017-10-03 | 2020-12-24 | キッコーマン株式会社 | アルカリホスファターゼの製造方法及びそれを用いて得られるアルカリホスファターゼ、並びにその製造のためのベクター及び形質転換体 |
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EP1460425A1 (en) | 2003-03-17 | 2004-09-22 | Boehringer Mannheim Gmbh | Deglycosylated enzymes for conjugates |
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EP1606619A1 (en) | 2005-12-21 |
CA2515686C (en) | 2011-03-29 |
CA2515686A1 (en) | 2004-09-30 |
JP2006520187A (ja) | 2006-09-07 |
DE602004007779T2 (de) | 2008-04-30 |
EP1460425A1 (en) | 2004-09-22 |
EP1606619B1 (en) | 2007-07-25 |
WO2004083862A1 (en) | 2004-09-30 |
US7482145B2 (en) | 2009-01-27 |
US20060040345A1 (en) | 2006-02-23 |
ATE368222T1 (de) | 2007-08-15 |
DE602004007779D1 (de) | 2007-09-06 |
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