JP6928668B2 - 増大した熱安定性を有する変異型逆転写酵素、ならびにそれに関する生成物、方法および使用 - Google Patents
増大した熱安定性を有する変異型逆転写酵素、ならびにそれに関する生成物、方法および使用 Download PDFInfo
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Description
i)配列番号1の野生型RTと比較して、
− 位置32のAlaがVal(A32V)で置換されており、
− 位置72のLeuがArg(L72R)で置換されており、
− 位置286のGluがArg(E286R)で置換されており、
− 位置302のGluがLys(E302K)で置換されており、
− 位置388のTrpがArg(W388R)で置換されており、
− 位置435のLeuがArg(L435R)で置換されている、
6個のアミノ酸置換を有するアミノ酸配列、または
ii)i)のアミノ酸配列と少なくとも95%同一でありi)で定義した6個のアミノ酸置換を有するアミノ酸配列を含み、
逆転写酵素活性を示す、変異型RTに関する。
− 位置32のAlaがVal(A32V)で置換されており、
− 位置72のLeuがArg(L72R)で置換されており、
− 位置286のGluがArg(E286R)で置換されており、
− 位置302のGluがLys(E302K)で置換されており、
− 位置388のTrpがArg(W388R)で置換されており、
− 位置435のLeuがArg(L435R)で置換されている、
6個の必須的アミノ酸置換がある。
Ala、Leu、Val、Ile 他の脂肪族残基(Ala、Leu、Val、Ile)
他の無極性残基(Ala、Leu、Val、Ile、Gly、Met)
Gly、Met 他の無極性残基(Ala、Leu、Val、Ile、Gly、Met)
Asp、Glu 他の酸性残基(Asp、Glu)
Lys、Arg 他の塩基性残基(Lys、Arg)
Asn、Gln、Ser、Thr 他の極性残基(Asn、Gln、Ser、Thr)
His、Tyr、Trp、Phe 他の芳香族残基(His、Tyr、Trp、Phe)
Cys、Pro なし
a)変異型RTの活性に資する条件下で、試料を本発明の変異型RTと接触させる工程、
b)本発明の変異型RTを使用してRNAマーカーからcDNAを合成する工程、および
c)ステップb)で合成したcDNAの存在を検出し、それによって試料中のRNAマーカーを検出する工程
を含む方法をさらに提供する。
− 細菌:ストレプトコッカス、スタフィロコッカス、シュードモナス、バークホルデリア、マイコバクテリウム、クラミドフィラ、エーリキア、リケッチア、サルモネラ、ナイセリア、ブルセラ、マイコバクテリウム、ノカルジア、リステリア、フランシセラ、レジオネラ、およびエルシニア
− ウイルス:アデノウイルス、単純ヘルペスウイルス、水痘帯状ヘルペスウイルス、サイトメガロウイルス、パピローマウイルス、B型肝炎ウイルス、C型肝炎ウイルス、E型肝炎ウイルス、ポリオウイルス、黄熱ウイルス、デング熱ウイルス、西ナイルウイルス、TBEウイルス、HIV、インフルエンザウイルス、ラッサウイルス、ロタウイルスおよびエボラウイルス
− 真菌:カンジダ、アスペルギルス、クリプトコッカス、ヒストプラズマ、ニューモシスティスおよびスタキボトリス
− 寄生体:原生動物寄生体、ぜん虫寄生体および節足動物寄生体。
RT濃度および標準RNA
標準としてウシ血清アルブミン(ナカライテスク株式会社、京都、日本)を含むプロテインアッセイCBB溶液(ナカライテスク株式会社)を使用して、Bradford(Bradford、1976年)の方法に従いRT濃度を決定した。バチルス・セレウス(Bacillus cereus)のcesA遺伝子のDNA配列8353〜9366(GenBank受託番号DQ360825)に相当する1014ヌクレオチドのRNAであった標準RNAは、in vitro転写によって調製した。
MMLV RT変異体の発現プラスミドを、野生型MMLV RT、pET−MRTHis用の発現プラスミド(図2)、または鋳型として熱的安定性変異体MM3、pET−MM3、および宿主として大腸菌BL21(DE3)[F−、ompT、hsdSB(rB −mB −)galdcm(DE3)]を使用して、部位特異的変異誘発によって構築した。変異型MMLV RT遺伝子のヌクレオチド配列を立証した。
50μg/ml アンピシリンを含有する3mlのLブロスを、形質転換BL21(DE3)のグリセロールストックに接種し、30℃で攪拌しながら16時間インキュベートした。RT遺伝子の発現は、自動誘導システム(Novagen、Darmstadt、ドイツ)によって誘導した。MMLV RTは、HisLink Spinタンパク質精製システム(Promega、Madison、WI)を使用して培養培地から精製した。簡単に言うと、培養物へのFastBreak細胞溶解試薬、次にHisLinkタンパク質精製樹脂の添加によって細菌細胞を破壊した。次いで試料をHisLink Spinカラムに移し、そこで非結合タンパク質を洗浄除去した。0.2mlの100mM HEPES−NaOHバッファー(pH7.5)、500mMイミダゾールを用いた溶出によってMMLV RTを回収した。50mMトリス−HClバッファー(pH8.3)、200mM KCl、50%グリセロールで平衡化した事前充填PD−10ゲル濾過カラム(GE Healthcare)を使用して活性分画を脱塩処理し、−80℃で保存した。
形質転換体の一晩培養物(5mL)を、アンピシリン(50μg/ml)を含有する500mLのLブロスに加え、37℃において攪拌しながらインキュベートした。OD660が0.6〜0.8に達したとき、0.15mLの0.5M IPTGを加え、3時間30℃で増殖させ続けた。10,000×gで5分間の遠心分離後、細胞を採取し、10mMフッ化フェニルメチルスルホニル(PMSF)、pH7.5を含有する10mLの0.02Mリン酸カリウムバッファー(pH7.2)、2.0mMジチオスレイトール(DTT)、10%グリセロール(バッファーA)で懸濁し、超音波処理によって破壊した。20,000×gで40分間の遠心分離後、上清を回収し、バッファーAで平衡化したToyopearl DEAE−650Mゲル(東ソー株式会社、東京、日本)充填カラム[25mm(内径)×120mm]に施した。120mM NaClを含有するバッファーAで洗浄した後、300mM NaClを含有するバッファーAで結合RTを溶出した。固体(NH4)2SO4を、40%飽和の最終濃度まで溶出液(30mL)に加えた。溶液は5分間攪拌し、氷上に30分間放置した。4℃における20,000×gで30分間の遠心分離後、ペレットを回収し、100mM NaClを含有する10mLバッファーA中に溶解した。50mMトリス−HClバッファー(pH8.3)、200mM KCl、2mM DTT、10%グリセロール(バッファーB)で平衡化したNi2+−セファロース(HisTrap HP 1mL、GE Healthcare、Buckinghamshire、UK)充填カラムに溶液を施した。50mMイミダゾールを含有するバッファーBで洗浄した後、500mMイミダゾールを含有するバッファーBで結合RTを溶出した。50mMトリス−HClバッファー(pH8.3)、200mM KCl、50%グリセロールで平衡化した事前充填PD−10ゲル濾過カラムを使用して活性分画を脱塩処理し、−80℃で保存した。
還元条件下において10%ポリアクリルアミドゲル中でSDS−PAGEを実施した。2.5%の2−メルカプトエタノールを用いた100℃で10分間の処理によりタンパク質を還元し、次いでゲル上に施した。40mAの定電流を40分間施した。電気泳動後、クーマシーブリリアントブルーR−250でタンパク質を染色した。ウサギ筋肉ホスホリラーゼB(97.2kDa)、ウシ血清アルブミン(66.4kDa)、ニワトリ卵白オボアルブミン(44.3kDa)、およびウシ炭酸脱水酵素(29.0kDa)からなる分子量マーカーキットは、タカラバイオ株式会社(大津、日本)の製品であった。
(dT)15(Fasmac、東京、日本)とポリ(rA)(GE Healthcare、Buckinghamshire、UK)のアニーリングによってポリ(rA)−p(dT)15を調製した。反応は、25mMトリス−HClバッファー(pH8.3)、50mM KCl、2mM DTT、5mM MgCl2、25μMポリ(rA)−p(dT)15(この濃度はp(dT)15に基づいて表す)、0.2mM [3H]dTTP(1.85Bq/pmol)(GE Healthcare)、および5または10nM MMLV RT中で37℃において実施した。3分と6分にアリコート(20μl)を反応混合物から入手し、すぐにガラスフィルター上にスポットした。取り込まれなかった[3H]dTTPは、それぞれ10分間3回の冷却5%(w/v)トリクロロ酢酸(TCA)での洗浄、次に冷却95%エタノールでの1回の洗浄によって除去した。2.5mlのEcoscint H(National Diagnostics、Yorkshire、UK)で、乾燥フィルター上で保持された放射活性を計測した。初期反応速度は、[3H]dTTPの取り込みに関する時間行程から推定した。
EnzChek逆転写酵素アッセイキット(Thermo Fisher Scientific、Waltham、MA)を使用した。19mlの水を加えて1×TEバッファーを作製することにより20×TEバッファー(1ml)を希釈した。PicoGreen dsDNA定量化試薬(50μl)は、17.5mlの1×TEバッファーを加えてPicoGreen溶液を作製することによって希釈した。加熱不活性化において使用するためのポリ(rA)−p(dT)16は以下のように調製した。ポリ(rA)(3μl、100mMトリス−HClバッファー(pH8.1)、0.5mM EDTA中1mg/ml、約350塩基)とp(dT)16(3μl、100mMトリス−HClバッファー(pH8.1)、0.5mM EDTA中50μg/ml)を混合し、室温で1時間放置し、次に114μlのPDGT(0.01Mリン酸カリウムバッファー(pH7.6)、2mM DTT、10%グリセロール、0.2%TritonX−100)で希釈した。MMLV RT(500nMの8μl)、ポリ(rA)−p(dT)16(8μl)、およびPDGT(64μl)を混合して、MMLV RT濃度を50nMにした。生成した溶液(80μl中40μl)を49℃または51℃で10分間インキュベートし、次に氷上で30分間インキュベートした。
バチルス・セレウスのcesA遺伝子のDNA配列8,353〜9,366(GenBank受託番号DQ360825)に相当する1,014ヌクレオチドのRNAである標準RNAは、in vitro転写によって調製した。cDNA合成用の反応混合物(20μl)は、水(12μl)、10×RTバッファー(250mMトリス−HClバッファー(pH8.3)、500mM KCl、20mM DTT、50mM MgCl2)(2μl)、2.0mM dNTP(1μl)、160pg/μl cesA RNA(2μl)、10μM RV−R26プライマー5’−TGTGGAATTGTGAGCGGTGTCGCAATCACCGTAACACGACGTAG−3’(配列番号4)(1μl)および10nM MMLV RT野生型(2μl)を混合することによって調製した。反応は45℃で30分間、および65℃で5分間実施した。PCR用の反応混合物(25μl)は、cDNA合成の反応産物(2μl)、水(17.7μl)、10×PCRバッファー(2.5μl)、2mM dNTP(1.5μl)、10μM F5プライマー5’−TGCGCGCAAAATGGGTATCAC−3’(配列番号5)(0.5μl)および10μM RVプライマー5’−TGTGGAATTGTGAGCGG−3’(配列番号6)(0.5μl)、およびTaqポリメラーゼ(0.3μl)を混合することによって調製した。反応は、95℃で30秒、55℃で30秒、および72℃で60秒の30サイクル下で実施した。増幅産物は1.0%w/vアガロースゲル上に分離させ、臭化エチジウム(1μg/ml)で染色した。
変異の設計および一重変異体の特徴付け
本発明者らは、鋳型−プライマーとの相互作用に関与している位置への正電荷の導入(Yasukawaら、2010年)によって、熱的安定性三重MMLV RT変異体MM3(E286R/E302K/L435R)を以前に作製した。MMLV RTをさらに安定化するため、本発明者らは29個の変異を設計した(表1)。それらは、疎水性コアを安定化する目的の8個の変異、塩架橋を導入する目的の8個の変異、表面電荷を導入する目的の10個の変異、およびジスルフィド結合を排除する目的の3個の変異である。
変異の組合せの設計および多重変異体の特徴付け
図3中に表した結果に基づき、4個の変異(Ala32→Val、Leu72→Arg、Ile212→Arg、Leu272→Glu、およびTrp388→Arg)を安定化変異として選択し、1個の変異(Leu41→Asp)を活性化変異として選択した。10個の変異体(MM3.1〜MM3.10)を、6個の変異中1、2、または3個とMM3変異(Glu286→Arg、Glu302→Lys、およびLeu435→Arg)を組み合わせることによって設計した(表2)。
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Kotewicz,M.L., D’Alessio,J.M., Driftmier,K.M., Blodgett,K.P. and Gerard,G.F. (1985) Gene, 35, 249-258.
Mizuno,M., Yasukawa,K. and Inouye,K. (2010) Biosci. Biotechnol. Biochem., 74, 440-442.
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Claims (15)
- 配列番号1の野生型逆転写酵素(RT)と比較して増大した熱安定性を有する変異型RTであって、
i)配列番号1の野生型RTと比較して、
− 位置32のAlaがVal(A32V)で置換され、
− 位置72のLeuがArg(L72R)で置換され、
− 位置286のGluがArg(E286R)で置換され、
− 位置302のGluがLys(E302K)で置換され、
− 位置388のTrpがArg(W388R)で置換され、および、
− 位置435のLeuがArg(L435R)で置換されている、
6個のアミノ酸置換を有するアミノ酸配列、または、
ii)i)のアミノ酸配列と少なくとも95%同一であり、i)で定義した6個のアミノ酸置換を有するアミノ酸配列、
を含み、
逆転写酵素活性を示し、
変異型MM3と比較して増大した熱安定性を有し、
MM3が位置286のGluがArg(E286R)で置換され、位置302のGluがLys(E302K)で置換され、および位置435のLeuがArg(L435R)で置換されている、3個のアミノ酸置換のみが配列番号1のアミノ酸配列と異なるアミノ酸配列を有する、
前記変異型RT。 - 追加的に、配列番号1のN末端における最大5個のアミノ酸の欠失、および/または配列番号1のC末端における最大5個のアミノ酸の欠失が、配列番号1のアミノ酸配列と異なる、請求項1に記載の変異型RT。
- 配列番号2のアミノ酸配列と少なくとも96%、97%、98%、99%、または100%同一であるアミノ酸配列を含むかまたはそれからなる、請求項1または2に記載の変異型RT。
- 熱安定性が、熱処理後に測定される変異体の逆転写酵素活性の測定によって決定される、請求項1〜3のいずれか一項に記載の変異体。
- 熱安定性が、60℃で10分間のインキュベーション後の逆転写酵素活性の測定によって決定され、および/または野生型もしくは変異型MM3と比較して少なくとも10%、20%、30%、40%、もしくは50%熱安定性が増大している、請求項1〜3のいずれか一項に記載の変異体。
- 変異型RTの逆転写酵素活性が、野生型の逆転写酵素活性の少なくとも50%であり、および/または逆転写酵素活性が、37℃でのRT媒介dTTP取り込みによって決定される、請求項1〜5のいずれか一項に記載の変異体。
- さらなるタンパク質と融合した、請求項1〜6のいずれか一項に記載の変異型RT。
- 請求項1〜7のいずれか一項に記載の変異型RTをコードする核酸。
- 請求項1〜7のいずれか一項に記載の変異型RTまたは請求項8に記載の核酸を含む細胞。
- 請求項1〜7のいずれか一項に記載の変異型RTを含む、逆転写を実施するためのキット。
- cDNA合成のための、請求項1〜7のいずれか一項に記載の変異型RTの使用。
- RNAを逆転写するための方法であって、請求項1〜7のいずれか一項に記載の変異型RTを使用してRNAからcDNAを合成する工程を含む方法。
- 試料中のRNAマーカーを検出するための方法であって、
a)変異型RTの活性に資する条件下で、試料を請求項1〜7のいずれか一項に記載の変異型RTと接触させる工程、
b)変異型RTを使用してRNAマーカーからcDNAを合成する工程、および、
c)ステップb)で合成したcDNAの存在を検出し、それによって試料中のRNAマーカーを検出する工程、
を含む、前記方法。 - 試料が、体液、血液、血漿、血清、尿、胆汁、脳脊髄液、スワブ、臨床検体、臓器試料および組織試料からなる群から選択され、ならびに/または試料が、細胞培養物、汚染の疑いがある供給源、もしくは対象から得られたものである、請求項13に記載の方法。
- RNAマーカーが、微生物、細胞、ウイルス、細菌、真菌、哺乳動物種、遺伝子状態または疾患を示す、請求項13または14に記載の方法。
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US20220290112A1 (en) * | 2019-07-26 | 2022-09-15 | Toyobo Co., Ltd. | Variant reverse transcriptase exhibiting excellent stability |
EP3805380A1 (en) * | 2019-10-07 | 2021-04-14 | Qiagen Beverly, LLC | Improved thermostable viral reverse transcriptase |
CN112795549B (zh) * | 2019-11-13 | 2023-11-28 | 广州达安基因股份有限公司 | 一种逆转录酶突变体 |
AU2021267940A1 (en) | 2020-05-08 | 2022-12-08 | President And Fellows Of Harvard College | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
CN114591930B (zh) * | 2022-05-10 | 2022-08-12 | 翌圣生物科技(上海)股份有限公司 | 增强型逆转录酶、编码dna、试剂盒及其在rna文库构建中的应用 |
CN117106745A (zh) * | 2022-06-01 | 2023-11-24 | 北京擎科生物科技股份有限公司 | 逆转录突变体及其应用 |
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