JP2013532958A - 増強された検出特性を有する変異プロテアーゼバイオセンサー - Google Patents
増強された検出特性を有する変異プロテアーゼバイオセンサー Download PDFInfo
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- JP2013532958A JP2013532958A JP2013510280A JP2013510280A JP2013532958A JP 2013532958 A JP2013532958 A JP 2013532958A JP 2013510280 A JP2013510280 A JP 2013510280A JP 2013510280 A JP2013510280 A JP 2013510280A JP 2013532958 A JP2013532958 A JP 2013532958A
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- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
- C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
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Abstract
Description
本出願は、2010年5月11日に提出された米国特許仮出願第61/333,706号及び2011年4月11日に提出された米国特許仮出願第61/470,845号に対する優先種を主張し、その個々は参照によりその全体が本明細書に組み込まれる。
配列記載書は、単に電子書式で本出願と共に提出され、そして参照により本明細書に組み込まれる。配列記載書テキストファイル「ASFILED_Sequence_WOoo.txt」は、2011年5月11日に作られ、そして152,614バイトのサイズである。
表1:対応するTL−CP358−DEVD:DDに対するCBS変異体の応答の改良倍率(fold improvement)の要約
表2.対応するTL−CP358−DEVD:DDに対する特定アミノ酸置換の組合せを有するCBS変異体の応答の改良性の要約
表3.クローン01:1−05、FC7:24、FC7:43 及び FC7:43に見出されるアミノ酸置換の要約
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Claims (70)
- 修飾された円順列熱安定性ルシフェラーゼ及び前記熱安定性ルシフェラーゼのN末端部分に前記熱安定性ルシフェラーゼのC末端部分を連結するリンカーを含むバイオセンサーポリペプチドをコードするポリヌクレオチドであって、前記修飾された円順列熱安定性ルシフェラーゼは親の円順列熱安定性ルシフェラーゼに関連して修飾され、前記リンカーは細胞中の標的分子と相互反応できるセンサー領域を含み、ここで前記修飾された円順列熱安定性ルシフェラーゼは、i)前記標的分子の存在下での前記親の円順列熱安定性ルシフェラーゼ、又はii)前記標的分子の不在下での前記修飾された円順列熱安定性ルシフェラーゼの少なくとも1つに関し、標的分子とバイオセンサーとの相互反応の後、増強された応答を有する、ポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2の位置507に対応するアミノ酸で置換を含む、請求項1記載のポリヌクレオチド。
- 前記位置507でのアミノ酸がIである、請求項2記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2の位置503に対応するアミノ酸で置換を含む、請求項1〜3のいずれか1項記載のポリヌクレオチド。
- 前記位置503でのアミノ酸がGである、請求項4記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2の位置471に対応するアミノ酸で置換を含む、請求項1〜5のいずれか1項記載のポリヌクレオチド。
- 前記位置471でのアミノ酸がTである、請求項6記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2の位置193に対応するアミノ酸で置換を含む、請求項1〜7のいずれか1項記載のポリヌクレオチド。
- 前記位置193でのアミノ酸がPである、請求項8記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2のI471T、 S503G、T507I及びS193Pに対応するアミノ酸置換を含む、請求項1〜9のいずれか1項記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2のI471T、S503G及びT507Iに対応するアミノ酸置換を含む、請求項1〜10のいずれか1項記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2のS503G、T507I及びS193Pに対応するアミノ酸置換を含む、請求項1〜11のいずれか1項記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2のT507Iに対応するアミノ酸置換を含む、請求項1〜12のいずれか1項記載のポリヌクレオチド。
- 前記修飾された円順列熱安定性ルシフェラーゼが、配列番号2の位置5、17、21、23、26、39、44、51、81、101、103、110、114、115、119、123、126、128、133、137、186、191、192、193、196、208、211、214、226、228、230、233、264、273、275、286、287、294、295、297、302、303、304、306、308、309、313、324、329、331、343、348、353、364、374、385、389、409、420、426、427、428、431、449、456、460、461、465、466、468、471、473、482、484、485、489、493、494、497、503、507、509、510、513、516、517、521、522、523、526、530、533、536、537、542又は543、又はそれらの組合せに対応する少なくとも1つのアミノ酸の置換を含む、請求項1〜13のいずれか1項記載のポリヌクレオチド。
- 前記熱安定性ルシフェラーゼが、ホタルルシフェラーゼの残基2−12、残基32−53、残基70−88、残基102−126、残基139−165、残基183−203、残基220−247、残基262−273、残基303−313、残基353−408、残基485−495又は残基535−546に対応する領域で円順列される、請求項1〜14のいずれか1項記載のポリヌクレオチド。
- 前記バイオセンサーが、プロテアーゼ認識部位、キナーゼ認識部位、抗体結合部位、金属結合部位、イオン結合部位、環状ヌクレオチド結合部位又はヌクレオチド結合部位を含む、請求項1〜15のいずれか1項記載のポリヌクレオチド。
- 前記バイオセンサーがプロテアーゼ認識部位を含む、請求項1〜16のいずれか1項記載のポリヌクレオチド。
- 前記プロテアーゼ認識部位が、カスパーゼ3認識部位、カスパーゼ8認識部位、エンテロキナーゼ認識部位、前立腺血清抗原認識部位、SARSウィルスプロテアーゼ認識部位、TEVプロテアーゼ認識部位、グランザイムB認識部位、MMP認識部位及びライノウィルスプロテアーゼ認識部位から成る群から選択される、請求項17記載のポリヌクレオチド。
- 前記熱安定性ルシフェラーゼが、ルキオラ・クルキアタ(Luciola cruciata)、ルキオラ・ラテラリス(Luciola lateralis)、ピロコエリア・ミヤコ(pyrocoelia miyako)、ラムピリス・ノクチルカ(Lampyris noctiluca)、フォツリス・ペンシルバニカ(Photuris pennsylvanica)、フェンゴデスsp.(Phengodes sp.)、ルキオラ・ミングレリカ(Luciola mingrelica)及びフォチナス・ピラリス(photinus pyralis)から成る群から選択された種からの野生型ルシフェラーゼから変性される、請求項1〜18のいずれか1項記載のポリヌクレオチド。
- 前記熱安定性ルシフェラーゼが、ホタルルシフェラーゼである、請求項1〜19のいずれか1項記載のポリヌクレオチド。
- 前記熱安定性ルシフェラーゼが、配列番号2に対して少なくとも95%のアミノ酸同一性を有する、請求項20記載のポリヌクレオチド。
- 前記熱安定性ルシフェラーゼが、配列番号4に対して少なくとも90%のアミノ酸同一性を有する、請求項20記載のポリヌクレオチド。
- 前記熱安定性ルシフェラーゼが、配列番号4に対して少なくとも95%のアミノ酸同一性を有する、請求項20記載のポリヌクレオチド。
- 前記リンカーが前記センサー領域DEVDを含む、請求項1〜23のいずれか1項記載のポリヌクレオチド。
- 前記リンカーが、SSDEVDGSSG(配列番号52)、SSGSDEVDGSLSSG(配列番号53)、SDEVDGSL(配列番号54)又はDEVDG(配列番号55)である、請求項1〜24のいずれか1項記載のポリヌクレオチド。
- 配列番号6のポリペプチドをコードするポリヌクレオチド。
- 請求項1〜26のいずれか1項記載のポリヌクレオチドを含むベクター。
- 前記ポリヌクレオチドがプロモーターに操作可能に結合される、請求項27記載のポリヌクレオチド。
- 請求項1〜26のいずれか1項記載のポリヌクレオチド、又は請求項27又は28記載のベクターを含む細胞。
- 請求項29記載の細胞を含む非ヒトトランスジェニック動物。
- 請求項1〜26のいずれか1項記載のポリヌクレオチドによりコードされる修飾された熱安定性ルシフェラーゼバイオセンサー。
- 修飾された熱安定性ルシフェラーゼバイオセンサーの発現を可能にする条件下で請求項29記載の細胞を増殖することを含む、修飾された熱安定性ルシフェラーゼバイオセンサーの製造方法。
- 修飾された熱安定性ルシフェラーゼバイオセンサーの発現を可能にする条件下で請求項27又は28記載のベクターを細胞中に導入することを含む、修飾された熱安定性ルシフェラーゼバイオセンサーの製造方法。
- 請求項1〜26のいずれか1項記載のポリヌクレオチド又は請求項27又は28記載のベクターを含むキット。
- 細胞中の標的分子の存在の検出方法であって、
a)前記細胞と、請求項1〜26のいずれか1項記載のポリヌクレオチド又は請求項27又は28記載のベクター及び修飾された熱安定性ルシフェラーゼのための基質とを接触させ;そして
b)前記細胞中のルミネッセンスを検出することを含み、この際、前記標的分子が前記修飾された熱安定性ルシフェラーゼと相互反応できる、方法。 - 細胞中の標的分子の存在の検出方法であって、
a)前記細胞と、請求項31記載の修飾された熱安定性ルシフェラーゼバイオセンサー及び前記修飾された熱安定性ルシフェラーゼのための基質とを接触させ;そして
b)前記細胞中のルミネッセンスを検出することを含み、この際、前記標的分子が前記修飾された熱安定性ルシフェラーゼバイオセンサーと相互反応できる、方法。 - サンプル中の標的分子の存在又は活性の検出方法であって、
a)標的分子のためのセンサー領域を含む修飾された円順列熱安定性ルシフェラーゼバイオセンサーをコードするポリヌクレオチド及び前記修飾された円順列熱安定性ルシフェラーゼのための基質と前記サンプルとを接触させ;そして
b)前記サンプル中のルミネッセンスを検出することを含む方法。 - 前記サンプルが、細胞、動物、細胞溶解物、又はin vitro転写/翻訳混合物である、請求項37記載の方法。
- 前記標的分子がプロテアーゼである、請求項35〜38のいずれか1項記載の方法。
- 前記プロテアーゼが、カスパーゼ3、カスパーゼ8、TEVプロテアーゼ又はMMP−2である、請求項39記載の方法。
- 前記標的分子の活性を変更できる試験化合物をさらに添加することを含む、請求項35〜40のいずれか1項記載の方法。
- ルミネッセンスがin vivoイメージングにより検出される、請求項35〜41のいずれか1項記載の方法。
- 前記修飾された円順列熱安定性ルシフェラーゼバイオセンサーが細胞において発現される、請求項37〜42のいずれか1項記載の方法。
- 前記修飾された円順列熱安定性ルシフェラーゼバイオセンサーを発現する細胞が、動物中に注入されるか又は移植される、請求項43記載の方法。
- 細胞中の標的分子の存在又は活性の検出方法であって、
a)標的分子のためのセンサー領域を含む修飾された円順列熱安定性ルシフェラーゼバイオセンサー及び前記修飾された円順列熱安定性ルシフェラーゼのための基質と細胞とを接触させ;そして
b)前記細胞中のルミネセンスを検出することを含む方法。 - 前記標的分子がプロテアーゼである、請求項45記載の方法。
- 前記プロテアーゼが、カスパーゼ3、カスパーゼ8、TEVプロテアーゼ又はMMP−2である、請求項46記載の方法。
- 前記標的分子の活性を変更できる試験化合物をさらに添加することを含む、請求項45〜47のいずれか1項記載の方法。
- 動物中の標的分子の存在又は活性の検出方法であって、
a)前記標的分子のためのセンサー領域を含む修飾された円順列熱安定性ルシフェラーゼバイオセンサー及び前記修飾された円順列熱安定性ルシフェラーゼのための基質と前記動物とを接触させ;そして
b)前記動物中のルミネッセンスを検出することを含む方法。 - 前記修飾された円順列熱安定性ルシフェラーゼバイオセンサーが細胞において発現される、請求項49記載の方法。
- 前記細胞が、前記動物中に注入されるか又は移植される、請求項50記載の方法。
- 前記ルシフェラーゼのための基質が前記動物中に注入される、請求項49〜51のいずれか1項記載の方法。
- 前記動物がマウスである、請求項49〜52のいずれか1項記載の方法。
- 前記標的分子の活性を変更できる試験化合物をさらに添加することを含む、請求項49〜53のいずれか1項記載の方法。
- ルミネッセンスがイメージングにより検出される、請求項49〜54のいずれか1項記載の方法。
- サンプル中の標的分子の存在又は活性の検出方法であって、
a)前記標的分子のためのセンサー領域を含む修飾された円順列熱安定性ルシフェラーゼバイオセンサーを、固体支持体上の固定し;
b)前記標的分子を含むサンプルを、前記固定されたバイオセンサーに添加し;
c)前記修飾された円順列熱安定性ルシフェラーゼのための基質を添加し、そして
d)ルミネッセンスを検出することを含む方法。 - 前記固体支持体が、粒子、樹脂、カラム、ウェルボトム、プレート又はスライドである、請求項56記載の方法。
- 前記標的分子がプロテアーゼである、請求項56又は57記載の方法。
- 前記プロテアーゼが、カスパーゼ3、カスパーゼ8、TEVプロテアーゼ又はMMP−2である、請求項58記載の方法。
- 前記修飾された円順列熱安定性ルシフェラーゼバイオセンサーが、精製されるか、又は細胞溶解物において発現される、請求項56〜59のいずれか1項記載の方法。
- 前記標的分子が精製されるか、又は細胞溶解物において発現される、請求項56〜60のいずれか1項記載の方法。
- サンプル中のアポトーシスの検出方法であって、
a)アポトーシスに関与する分子のためのセンサー領域を含む修飾された円順列熱安定性ルシフェラーゼバイオセンサーをコードするポリヌクレオチド及び前記修飾された円順列熱安定性ルシフェラーゼのための基質と前記サンプルとを接触させ;そして
b)前記サンプル中のルミネッセンスを検出することを含む方法。 - 前記サンプルが、細胞、動物、細胞溶解物、又はin vitro転写/翻訳混合物である、請求項62記載の方法。
- 前記標的分子がプロテアーゼである、請求項62又は63記載の方法。
- 前記プロテアーゼが、カスパーゼ3又はカスパーゼ8である、請求項64記載の方法。
- 前記標的分子の活性を変更できる試験化合物をさらに添加することを含む、請求項62〜65のいずれか1項記載の方法。
- ルミネッセンスがin vivoイメージングにより検出される、請求項62〜66のいずれか1項記載の方法。
- 前記修飾された円順列熱安定性ルシフェラーゼバイオセンサーが細胞において発現される、請求項62〜67のいずれか1項記載の方法。
- 前記修飾された円順列熱安定性ルシフェラーゼバイオセンサーを発現する細胞が、動物中に注入されるか又は移植される、請求項68記載の方法。
- 前記修飾された円順列熱安定性ルシフェラーゼバイオセンサーが、請求項1〜26のいずれか1項記載のポリヌクレオチドによりコードされる、請求項37〜69のいずれか1項記載の方法。
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US20140298500A1 (en) | 2014-10-02 |
US9339561B2 (en) | 2016-05-17 |
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US20140308211A1 (en) | 2014-10-16 |
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US8735559B2 (en) | 2014-05-27 |
EP3508570B1 (en) | 2020-07-22 |
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US20110283373A1 (en) | 2011-11-17 |
US9248201B2 (en) | 2016-02-02 |
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