JP6282270B2 - 組み換えにより生成されたパエニバシラス・ポリミキサに由来する中性プロテアーゼ - Google Patents
組み換えにより生成されたパエニバシラス・ポリミキサに由来する中性プロテアーゼ Download PDFInfo
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- JP6282270B2 JP6282270B2 JP2015520928A JP2015520928A JP6282270B2 JP 6282270 B2 JP6282270 B2 JP 6282270B2 JP 2015520928 A JP2015520928 A JP 2015520928A JP 2015520928 A JP2015520928 A JP 2015520928A JP 6282270 B2 JP6282270 B2 JP 6282270B2
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Description
本明細書で開示されるような全ての態様の第9観点は、少なくとも1種類の本明細書で開示されるようなベクターを含有する形質転換された原核グラム陽性宿主生物である。
発現コンストラクト(DNA)の構築
ポリメラーゼ連鎖反応(PCR)ならびに所望のコードおよび非コードゲノムDNA鎖の部分配列(subsequences)に相当するいくつかの合成された一本鎖および/または二本鎖DNAオリゴヌクレオチドを用いて、人工遺伝子配列が生成された。まず第一に、向かい合う鎖の断片に相当する部分的に重複しているオリゴヌクレオチドの対を鋳型DNAとハイブリダイズさせ、二本鎖DNA分子をポリメラーゼに媒介される鎖の伸長により生成し、続いてPCR増幅を行った。さらなるDNA分子を合成により作製した。人工的に生成されたDNAの全ての配列を配列決定により検証した。
公開された配列情報を用いた発現コンストラクト
P.ポリミキサの中性プロテアーゼを発現させるための最初の試みは、Takekawa, S., et al., J. Bacteriology 173 (1991) 6820-6825に基づいていた。第1段階として、SEQ ID NO:1のヌクレオチド配列、特にSEQ ID NO:2をコードするオープンリーディングフレームに対応するCDSの部分配列(343)..(2115)を、コドン使用頻度を変化させることにより適合させた。コードされるアミノ酸配列は変化しないままであったが、オープンリーディングフレームをバシラス・サチリス(Bacillus subtilis)中での発現のために最適化する中立変異が導入された。SEQ ID NO:2をコードするリーディングフレームを有する人工DNAを作製し、合成した。それはP.ポリミキサの590アミノ酸を有するプレプロ酵素のアミノ酸配列、すなわちシグナル配列およびプロペプチドが含まれるアミノ酸配列をコードしていた。その発現コンストラクトにおいて、B.サチリス特異的リボソーム結合部位がそのオープンリーディングフレームの上流に導入された。そのDNAコンストラクトを、B.サチリスにおいて液体培地中での増殖の静止期において増殖期特異的プロモーターに駆動される転写を提供する発現ベクター中にクローニングした。結果として得られた選択可能かつ複製能力のある発現プラスミドはpLE2D01nprPpであった。
パエニバシラス・ポリミキサ株ATCC 21993に関する配列決定の結果
パエニバシラス・ポリミキサ株ATCC 21993から単離された総ゲノムDNAを単離し、その中性プロテアーゼをコードする遺伝子をPCRを用いて増幅した。その増幅されたDNAを配列決定した。驚くべきことに、DNA配列レベルでのいくつかの違いが見付かり、その違いはコードされるアミノ酸配列における変化をもたらしている。ATCC 21993株の中性プロテアーゼ遺伝子のアミノ酸配列をSEQ ID NO:5において示す。
公開された配列情報を用いた発現コンストラクト
その中性プロテアーゼをコードするDNAを、実施例3において記載されるようにパエニバシラス・ポリミキサATCC 21993株から単離した。実施例2に類似して、SEQ ID NO:5のアミノ酸配列に基づいて、その中性プロテアーゼをコードするB.サチリスにおける発現のためのDNA配列を異なる発現ベクター中に考案(devised)およびクローニングした。前記のパエニバシラス・ポリミキサATCC 21993株の中性プロテアーゼ(プレプロ酵素)のコード配列が含まれるクローニングされた断片のDNA配列を、SEQ ID NO:6として示す。典型的なコンストラクトは、そのシグナル配列およびそのプロペプチドが含まれるプレプロ酵素のP.ポリミキサのアミノ酸配列をコードしていた。そのDNAコンストラクトを、液体培地中での増殖の静止期におけるB.サチリス中での転写を駆動する増殖期特異的プロモーターを提供する発現ベクター中でクローニングする。結果として得られた選択可能かつ複製能力のある発現プラスミドはpLE2D01DisnatPpであった。
液体培地中のタンパク質分解活性の決定
EnzChek(登録商標)プロテアーゼアッセイキットを用いた(Invitrogen、E6638)。その直接的な蛍光に基づくアッセイは、金属プロテアーゼ、セリンプロテアーゼ、酸性プロテアーゼおよびスルフヒドリルプロテアーゼを検出する。そのアッセイキットは、pH非感受性緑色蛍光性BODIPY(登録商標)FL(E6638)色素で標識されているカゼイン誘導体を含有し、それは結果としてそのコンジュゲートの蛍光のほとんど完全な消光をもたらす。プロテアーゼに触媒される加水分解は蛍光性BODIPY FL色素で標識されたペプチドを遊離させる。分光蛍光計、ミニ蛍光計またはマイクロプレートリーダーを用いて測定することができる蛍光におけるそれに伴う増大は、プロテアーゼ活性に比例している。
Claims (7)
- 中性プロテアーゼを組み換えにより生成するための方法であって、以下の工程:
(a)発現ベクター中でSEQ ID NO:5に従うプレプロ酵素をコードしている配列を有するDNAを提供し、宿主生物のバシラス・アミロリケファシエンスの種を該発現ベクターを用いて形質転換し、それにより形質転換された宿主生物を得て;続いて
(b)該形質転換された宿主生物中で該DNAを発現させ、ここで該形質転換された宿主生物が該中性プロテアーゼを分泌し;続いて
(c)該分泌された中性プロテアーゼを単離する;
を含み、それにより該中性プロテアーゼを組み換えにより生成する、前記方法。 - DNAがSEQ ID NO:6の34位〜1896位の配列を含む、請求項1に記載の方法。
- 工程(b)が形質転換された宿主生物を液体培地中で培養することを含み、該形質転換された宿主生物が中性プロテアーゼを液体培地中に分泌する、請求項1または2に記載の方法。
- 工程(c)が分泌された中性プロテアーゼを液体培地から単離することを含む、請求項3に記載の方法。
- 宿主生物がNprおよびAprから選択される細胞外プロテアーゼを欠損している、請求項1〜4のいずれか1項に記載の方法。
- 少なくとも1種類のベクターを含有する、形質転換されたバシラス・アミロリケファシエンスの種である宿主生物であって、
ここで該ベクターはポリヌクレオチドを含有し、
該ポリヌクレオチドは、SEQ ID NO:5の289位〜592位のアミノ酸配列を含むポリペプチドをコードし、該ポリヌクレオチドは、以下:
(a)SEQ ID NO:6中の898位〜1811位の配列を有するポリヌクレオチド;
(b)縮重コードの結果としてのSEQ ID NO:6の898位〜1811位のヌクレオチド配列に由来するポリヌクレオチド;
からなる群から選択される、
前記宿主生物。 - SEQ ID NO:6の34位〜1811の配列を有する、請求項6に記載の宿主生物。
Applications Claiming Priority (3)
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EP12175563 | 2012-07-09 | ||
EP12175563.1 | 2012-07-09 | ||
PCT/EP2013/064271 WO2014009276A1 (en) | 2012-07-09 | 2013-07-05 | Recombinantly produced neutral protease originating from paenibacillus polymyxa |
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JP6282270B2 true JP6282270B2 (ja) | 2018-02-21 |
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JP2015520928A Active JP6282270B2 (ja) | 2012-07-09 | 2013-07-05 | 組み換えにより生成されたパエニバシラス・ポリミキサに由来する中性プロテアーゼ |
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US (5) | US20150166973A1 (ja) |
EP (1) | EP2870173B1 (ja) |
JP (1) | JP6282270B2 (ja) |
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WO2014009276A1 (en) * | 2012-07-09 | 2014-01-16 | Roche Diagnostics Gmbh | Recombinantly produced neutral protease originating from paenibacillus polymyxa |
US11146376B2 (en) | 2015-04-22 | 2021-10-12 | Qualcomm Incorporated | System type dependent master information block (MIB) |
US10334617B2 (en) * | 2015-06-16 | 2019-06-25 | Qualcomm Incorporated | System information for enhanced machine type communication |
US10384678B1 (en) | 2016-01-22 | 2019-08-20 | State Farm Mutual Automobile Insurance Company | Autonomous vehicle action communications |
KR101898662B1 (ko) * | 2017-06-01 | 2018-09-13 | 단국대학교 천안캠퍼스 산학협력단 | 바실러스 아밀로리퀘파시엔스 kj5 균주 및 이를 포함하는 친환경음식물쓰레기처리제제 |
CN114540221B (zh) * | 2022-01-19 | 2023-03-21 | 广东省科学院动物研究所 | 产中性蛋白酶芽孢杆菌和利用其生产中性蛋白酶的方法 |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3133001A (en) | 1959-11-26 | 1964-05-12 | Muset Pedro Puig | Stabilization of enzymes |
JPS5651747B2 (ja) * | 1973-05-31 | 1981-12-08 | ||
CA1133620A (en) | 1979-08-23 | 1982-10-12 | Patrick R. Beirne | Pbx field display |
US4304866A (en) * | 1979-11-14 | 1981-12-08 | Massachusetts Institute Of Technology | Transplantable sheets of living keratinous tissue |
US5762502A (en) * | 1996-07-11 | 1998-06-09 | Bahn; Arthur N. | Process for adhering composites to human teeth |
US5830741A (en) * | 1996-12-06 | 1998-11-03 | Boehringer Mannheim Corporation | Composition for tissue dissociation containing collagenase I and II from clostridium histolyticum and a neutral protease |
ES2597837T3 (es) * | 2003-06-27 | 2017-01-23 | DePuy Synthes Products, Inc. | Células posparto derivadas de tejido de la placenta, y métodos de fabricación y utilización de los mismos |
US20060269527A1 (en) * | 2005-05-26 | 2006-11-30 | Australian Stem Cell Centre Limited | Isolation of cells from bone |
US8673646B2 (en) * | 2008-05-13 | 2014-03-18 | General Atomics | Electrochemical biosensor for direct determination of percentage of glycated hemoglobin |
US8211684B2 (en) * | 2008-08-27 | 2012-07-03 | Roche Diagnostics Operations, Inc. | Stabilization of thermolysin in aqueous solution |
EP2589651A3 (en) * | 2008-11-11 | 2013-08-28 | Danisco US Inc. | Compositions and methods comprising serine protease variants |
WO2010105820A1 (en) | 2009-03-19 | 2010-09-23 | Roche Diagnostics Gmbh | Improved blends containing proteases |
CA2768562A1 (en) * | 2009-07-21 | 2011-01-27 | Transgene Sa | Enzymatic composition for the digestion of chicken embryos |
WO2014009276A1 (en) * | 2012-07-09 | 2014-01-16 | Roche Diagnostics Gmbh | Recombinantly produced neutral protease originating from paenibacillus polymyxa |
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US10526594B2 (en) | 2020-01-07 |
EP2870173A1 (en) | 2015-05-13 |
US20200115692A1 (en) | 2020-04-16 |
US10889809B2 (en) | 2021-01-12 |
JP2015521856A (ja) | 2015-08-03 |
EP2870173B1 (en) | 2018-10-17 |
US11542489B2 (en) | 2023-01-03 |
US20170037390A1 (en) | 2017-02-09 |
WO2014009276A1 (en) | 2014-01-16 |
US20210355472A1 (en) | 2021-11-18 |
ES2701763T3 (es) | 2019-02-25 |
US20150166973A1 (en) | 2015-06-18 |
US20190203191A1 (en) | 2019-07-04 |
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