JP5074768B2 - インビトロにおけるタンパク質合成の改良された方法 - Google Patents
インビトロにおけるタンパク質合成の改良された方法 Download PDFInfo
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- JP5074768B2 JP5074768B2 JP2006541404A JP2006541404A JP5074768B2 JP 5074768 B2 JP5074768 B2 JP 5074768B2 JP 2006541404 A JP2006541404 A JP 2006541404A JP 2006541404 A JP2006541404 A JP 2006541404A JP 5074768 B2 JP5074768 B2 JP 5074768B2
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Description
操作された生物学的高分子の合成は、生化学の多大な功績の一つである。組換えDNA(rDNA)技術の到来により、所望のタンパク質を作製するために細胞の触媒機構を活用することが可能となった。これは、細胞環境において、または細胞に由来する抽出物を使用してインビトロで達成され得る。
Swartz et al.、米国特許第6,337,191 B1号。Kim and Swartz(2000)Biotechnol Prog. 16: 385-390;Kim and Swartz(2000)Biotechnol Lett. 22:1537-1542;Kim and Choi (2000)J Biotechnol.84:27-32;Kim et al.(1996)Eur J Biochem.239:881-886;Kim and Swartz (2001)Biotechnol Bioeng 74:309-316;Davanloo et al., Proc Nat'l Acad Sci USA 81:2035-2039 (1984);Datsenko et al., Proc Nat'l Acad Sci USA 97:6640-6645(2000);「遺伝子クローニングおよび発現技術」(Gene Cloning and Expression Technologies)(Weiner,M.P. and Lu,Q.: eds.), Eaton Publishing, Westborough, MA. 391-411頁のJewett et al.(2002)「インビトロ発現のための原核生物系」(Prokaryotic systems for in vitro expression);Spirin et al., Science 242:1162-1164(1988)。
改良された収率、低下したコスト、および増強された利用可能性を提供する、生物学的分子のインビトロ合成のための改良された方法が提供される。改良された無細胞タンパク質合成反応は、例えば、グルコース、グルタミン酸、ピルビン酸等を含む、無リン酸エネルギー源をATP生成のために利用する。任意で、ヌクレオシド三リン酸がヌクレオシド一リン酸に交換される。これらの改良は、劇的にコストを減少させ、無細胞タンパク質合成反応の頑強さを増加させる。反応は、外因性リン酸の添加により実質的に改良される。
改良された収率、低下したコスト、および増強された利用可能性を提供する、生物学的高分子のインビトロ合成のための改良された方法が提供される。改良された収率および低下したコストは、以下に制限はされないが、無リン酸エネルギー源の使用、外因性ヌクレオシド三リン酸の欠如、ヌクレオシド一リン酸および外因性有機リン酸の存在を含む、反応条件の組み合わせにより入手される。
目的の合成系には、DNAの増幅、DNAまたはRNAの鋳型からのRNAの転写、RNAのポリペプチドへの翻訳、および単糖からの複合糖質の合成を含み得る、バイオポリマーの複製のための系が含まれる。増強された合成には、系中で合成されたポリペプチドの総量または相対量の増加;時間単位当たりに合成されたポリペプチドの総量または相対量の増加;系中で合成された生物学的に活性なポリペプチドの総量または相対量の増加;系中で合成された可溶性ポリペプチドの総量または相対量の増加等が含まれる。
実施例1
エネルギー源としてのグルコース
方法および材料
合成のための標準細胞模倣(Cytomim)環境は、以下の成分を含有している:1.2mM ATP、各0.85mMのGTP、UTP、およびCTP、1mM DTT、130mMグルタミン酸カリウム、10mMグルタミン酸アンモニウム、8 mMグルタミン酸マグネシウム、34μg/mlフォリン酸、170.6μg/ml大腸菌tRNA混合物、13.3μg/mlプラスミド、100μg/ml T7 RNAポリメラーゼ、各2mMの20種の未標識アミノ酸、11μM [14C]ロイシン、1.5mMスペルミジンおよび1mMプトレシン、0.33mMニコチンアミドアデニンジヌクレオチド、0.26mM補酵素A、2.7mMシュウ酸ナトリウム、および0.24倍容量のS30抽出物。原核生物無細胞タンパク質合成は、Pratt, J.M. 1984のプロトコル、「転写および翻訳:実際のアプローチ」(Transcription and translation:a practical approach), Hanes, B.D., and S.J. Higgins. (Eds.), IRL Press, New Yorkの179〜209頁の、「原核生物無細胞系における転写翻訳共役」(Coupled transcription-translation in prokaryotic cell-free systems)をわずかに改変した変法により、大腸菌K12(Michel-Reydellet et al.(2004)Met Eng(6)197-203に記載されたKC1株、遺伝子型A19ΔtonAΔtnaAΔspeAΔendAΔsdaAΔsdaB met+)に由来する粗製S30抽出物を使用して実施される。抽出物のための細胞は、規定培地で培養される(Saha B編、「発酵バイオテクノロジー」(Fermentation Biotechnology)(Washington, DC:ACS Press)の142-156頁のZawada et al.(2003)「大腸菌細胞抽出物の作製のための高密度の規定培地培養」(High-density, defined media culture for the production of Escherichia coli cell extracts))。T7 RNAポリメラーゼは、Davanloo et al. 1984、「バクテリオファージT7 RNAポリメラーゼの遺伝子のクローニングおよび発現」(Cloning and expression of the gene for bacteriophage T7 RNA polymerase)Proc Nat'l Acad. Sci.USA 81:2035-2039)の手法に従って、大腸菌BL21株(pAR1219)から調製された。33mMピルビン酸ナトリウムの添加は、必要ではないが、これによって系が増強され得る。各反応物中には、細胞抽出物に起因するおよそ3.3mMのマグネシウム、14.4mMのカリウム、2.4mMのトリス、および23.5mMの酢酸が、さらに存在した。
転写翻訳共役反応のための標準的な反応混合物は、Kim and Swartz(2001)により記載されている。標準的な反応におけるエネルギー源は、ホスホエノールピルビン酸(PEP)である。以前にPEPをグルコースに直接置換したところ、事実上タンパク質合成は生じなかった。しかしながら、PEPをグルコース-6-リン酸(G6P)に交換した場合、228±13μg/mLという有意なタンパク質収率が観察された(Kim and Swartz 2001)。グルコース-6-リン酸は解糖反応経路においてグルコースから一工程離れているだけであり、このことから、最初のリン酸化の工程が律速性であることが示唆される。ヘキソキナーゼもしくはグルコキナーゼの反応物への添加、または緩徐なグルコース投入を含む、いくつかの実験が、この律速を軽減するために実施された。いずれのアプローチも成功しなかった。
(表1)様々なエネルギー源およびヌクレオチドを用いた無細胞反応の比較
エネルギー源としてのグルタミン酸
下記の組み合わせられた転写−翻訳反応のための反応混合物は、以下の成分を含有している:1.2mM ATP、各0.85mMのGTP、UTP、およびCTP、130mMグルタミン酸カリウム、10mMグルタミン酸アンモニウム、10mMグルタミン酸マグネシウム、1.5mMスペルミジン、1mMプトレシン、34μg/mlフォリン酸、170.6μg/ml大腸菌tRNA混合物、13.3μg/mlプラスミド、100μg/ml T7 RNAポリメラーゼ、各2mMの20種の未標識アミノ酸、5μM L-[U-14C]-ロイシン、0.33mMニコチンアミド・アデニン・ジヌクレオチド、0.26mM補酵素A、4.0mMシュウ酸ナトリウム、および0.24倍容量のS30抽出物。
(表2)
表2 相対全CAT発現に基づく細胞模倣系におけるリン酸最適化研究。全ての実験が、ピルビン酸ナトリウムを含まずNTPを含む細胞模倣系からの条件を使用して実行された。15マイクロリットルの反応物が37℃で6時間インキュベートされた。氷酢酸によりpH7.25に調整されたリン酸カリウム(二塩基性、Mallinckrodt:Phillipsburg, NJ)が使用された。
表3 PANOx-SP系(NTP使用)、細胞模倣系(ピルビン酸なし、リン酸およびNTP使用)、および細胞模倣系(ピルビン酸なし、リン酸およびNMP使用)の産物収率(mgタンパク質/$エネルギー源およびヌクレオチドのコスト)。
Claims (16)
- 細菌細胞抽出物、ポリヌクレオチドおよび/またはポリペプチド作製のための鋳型、合成されるポリヌクレオチドおよび/またはポリペプチドのためのモノマー、および合成に必要な補因子、酵素、およびその他の試薬等を初めに含む、無細胞反応混合物における、ポリヌクレオチドおよび/またはポリペプチドを合成するための方法であって、
少なくとも10 mMの無リン酸エネルギー源、外因性ヌクレオシド三リン酸の非存在下においてヌクレオシド一リン酸、および少なくとも1 mMの濃度の外因性リン酸を含むように改変された無細胞反応混合物において、ポリヌクレオチドおよび/またはポリペプチドを合成する段階を含む、前記方法。 - 無リン酸エネルギー源がグルコースである、請求項1記載の方法。
- 無リン酸エネルギー源がグルタミン酸である、請求項1記載の方法。
- 無リン酸エネルギー源がピルビン酸である、請求項1記載の方法。
- リン酸が1mM〜20mMの濃度で存在する、請求項1記載の方法。
- リン酸が、リン酸カリウム、リン酸マグネシウム、またはリン酸アンモニウムとして提供される、請求項5記載の方法。
- 合成が、ポリペプチドを生成させるためのmRNAの翻訳を含む、請求項1記載の方法。
- 合成が、DNA鋳型からのmRNAの転写をも含む、請求項7記載の方法。
- ポリヌクレオチドおよび/またはポリペプチドの合成が回分反応として実施される、請求項1記載の方法。
- ポリヌクレオチドおよび/またはポリペプチドの合成が連続反応として実施される、請求項1記載の方法。
- 反応混合物がグルコース含有培地で培養された大腸菌からの抽出物を含む、請求項1記載の方法。
- 大腸菌が、グルコースおよびリン酸を含有する培地で培養される、請求項11記載の方法。
- 反応混合物が、5mM〜20mMの濃度のマグネシウムを含む、請求項1記載の方法。
- 反応混合物がポリエチレングリコールを含まない、請求項1記載の方法。
- 反応混合物が、スペルミン、スペルミジン、およびプトレシンのうちの一つまたは複数を含む、請求項14記載の方法。
- 反応混合物が、400μg/mLを超える合成されたポリペプチド収率を与える、請求項1記載の方法。
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PCT/US2004/038830 WO2005052117A2 (en) | 2003-11-20 | 2004-11-18 | Improved methods of in vitro protein synthesis |
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JP6280367B2 (ja) | 2010-08-31 | 2018-02-14 | グリーンライト バイオサイエンシーズ インコーポレーテッドGreenlight Biosciences,Inc. | プロテアーゼ操作を介した代謝経路におけるフラックスの制御のための方法 |
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KR20190103256A (ko) | 2016-12-30 | 2019-09-04 | 수트로박스, 인코포레이티드 | 비자연 아미노산을 갖는 폴리펩타이드-항원 접합체 |
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