JP2014506462A - 熱安定性が増加した逆転写酵素 - Google Patents
熱安定性が増加した逆転写酵素 Download PDFInfo
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- JP2014506462A JP2014506462A JP2013553352A JP2013553352A JP2014506462A JP 2014506462 A JP2014506462 A JP 2014506462A JP 2013553352 A JP2013553352 A JP 2013553352A JP 2013553352 A JP2013553352 A JP 2013553352A JP 2014506462 A JP2014506462 A JP 2014506462A
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Abstract
【選択図】図1
Description
配列番号1に記載されるアミノ酸配列を有するM-MLV由来の逆転写酵素に突然変異を導入するため、前記M-MLV由来の逆転写酵素をコーディングする遺伝子上の塩基配列を置き換えた。具体的に、配列番号1に記載されるアミノ酸配列を有するM-MLV由来の逆転写酵素で63番目のグルタミンからロイシンへの変異(Q63L)、264番目のリシンからロイシンへの変異(K264L)、295番目のリシンからグルタミンへの変異(K295Q)、306番目のトレオニンからロイシンへの変異(T306L)、346番目のグルタミン酸からメチオニンへの変異(E346M)、408番目のプロリンでグルタミン酸への変異(P408E)、438番目のヒスチジンからチロシンへの変異(H438Y変異)、及び454番目のアスパラギンからフェニルアラニンへの変異(N454F)が導入された8種の突然変異逆転写酵素を製作しており(図1を参照)、このため前記配列番号1のアミノ酸配列を有する逆転写酵素をコーディングする配列番号10に記載される遺伝子に突然変異を導入した(図2ないし図9を参照)。前記突然変異遺伝子は、それぞれ配列番号11ないし配列番号18に記載される塩基配列を有する。前記突然変異遺伝子の製造過程は、次の通りである:
大腸菌(Escherichia coli)でのタンパク質発現量を高めるため、配列番号10に記載されるM-MLV逆転写酵素の遺伝子配列を大腸菌のコドン利用性(codon usage)に合わせて最適化させ、配列番号19に記載される塩基配列を得た。前記最適化された塩基配列に基づき野生型遺伝子とK295Q突然変異遺伝子を合成した。先ず、配列番号19に記載される野生型遺伝子の塩基配列を得るため、全塩基配列に基づきセンス鎖とアンチセンス鎖をそれぞれアニーリング(annealing)温度が大凡60℃(20ないし40塩基)になるように設計したあと、設計されたオリゴヌクレオチド等を合成した(バイオニア、KOREA)。合成されたオリゴヌクレオチド等はそれぞれ配列番号20ないし配列番号149に記載される塩基配列を有する。前記合成されたオリゴヌクレオチド等を用いてキネーション(Kination)-LCR法(大韓民国特許出願第2008-0025050号)で40℃と60℃で反復的に反応させてライゲーション(ligation)させることにより、配列番号19に記載される塩基配列を有する二重鎖の全体遺伝子を合成した。さらに、K295Q突然変異遺伝子は、配列番号77及び配列番号78に記載される塩基配列の代りに配列番号150及び配列番号151に記載される塩基配列を用いたことを除いては、前記野生型遺伝子と同一の方法を利用して合成した。合成が完了した遺伝子を95℃で5分間予め熱処理したあと、配列番号152及び配列番号153に記載されるプライマー対を利用して95℃で1分、65℃で1分、72℃で2分30秒間反応させ、この過程を30回繰り返したあと72℃でさらに10分間反応させて前記遺伝子を増量した。前記増量された合成遺伝子をpGEM-T easy vector(Promega、USA)にライゲーションさせたあと、大腸菌DH5a(RBC、USA)に形質転換させて野生型及びK295Q突然変異遺伝子のクローンを多量確保した。
核外遺伝子PET 22b(+)(NOVAGEN CO.)を組換ベクターに用いて実施例1で製造された8種の突然変異逆転写酵素遺伝子をクローニングした。このため、pGEM-T easyベクター(Promega)に挿入して合成された変異遺伝子(Q63L、K264L、T306L及びE346M)を、下記プライマー対を利用してPCRを進めた。
アンチセンス鎖:5'-GCG CGC GCG GCC GCT TAG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3'(Not I)(配列番号169)
他の変異遺伝子(K295Q、P408E、H438Y、N454F)の場合、下記プライマー対を利用してPCRを進めた。
アンチセンス鎖:5'-GCG CGC GCG GCC GCG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3'(Not I)(配列番号171)
核外遺伝子ベクターPET 22b(+)とPCRとを介して得た各M-MLV逆転写遺伝子断片に対し、5'末端はNdeI、3'末端はNotIを処理して切断したあと、ゲル抽出キット(BIONEER CO.)を利用して精製した。以後、T4 DNAリガーゼ(ligase)(TAKARA CO.)を利用して前記切断された断片等を16℃で2時間反応させ、それぞれの突然変異遺伝子を核外遺伝子ベクターPET 22b(+)に挿入した。
T7ターミネーターアンチセンス鎖:5'-GCT AGT TAT TGC TCA GCG G-3'(配列番号173)
BL21(DE3)宿主細胞に形質転換した発現菌株の単一コロニーを採取したあと、アンピシリン(50mg/ml)が含まれたLB(Luria-Bertani medium)細胞培養液15mlに投入し、37℃で一晩中培養してシード(seed)菌株細胞を確保した。アンピシリンが含まれたLB細胞培養液1lに前記シード菌株細胞を投入して37℃で2時間の間培養したあと、0.5mM IPTG(Isopropyl β-D-1-thiogalactopyranoside)を添加して2時間さらに発現させた。培養されたそれぞれの菌株細胞を遠心分離したあと、得られたペレット(pellet)を-50℃で精製する前まで保管した。
実施例3で得られた凍結細胞塊5gを破砕したあと、4℃で攪拌しながら溶菌緩衝液(25mMトリス塩酸(Tris-HCl pH 7.6)、1mM EDTA、10%(v/v)β-メルカプトエタノール、フェニルメチルスルホニルフルオリド(PMSF)20ml)に30分間懸濁させた。その後、細胞均質器(fisher sonicator)で細胞を溶菌させた。その後、前記細胞溶菌物を4℃で1時間の間11,000rpmで遠心分離させ、ペレットを溢して捨てた。澄んだ溶菌物を直ちに緩衝溶液(40mMトリス塩酸(Tris-HCl pH 7.6)、2mM EDTA、28.58mM β-メルカプトエタノール、100mM KCl)に透析した。
突然変異逆転写酵素を精製するため、アマシャムFPLC装置を用いて行った。1次コラムはアマシャムXK16(1.6cm×30.0cm)を用いた。コラムにイオン交換樹脂コラムクロマトグラフィーDEAE hyper-D(pall co.)を充填し、1M KClを含有するコラム緩衝液250mlで洗浄したあと、緩衝溶液で平衡化させた60mlのベッドを得た。その後、透明な細胞溶菌物20mlを5ml/分の流速でコラムに適用した。適用された溶菌物のうちコラムに吸着されていない溶菌物を獲得して次のコラムに適用した。得られた溶菌物を2次クロマトグラフィーに適用した。コラムアマシャムXK16(1.6cm×30.0cm)を洗浄して平衡化させたあと、親和性クロマトグラフィーヘパリンhyper-D(pall co.)を充填して50mlのベッドを得た。その後、緩衝溶液で平衡化させた。溶菌物をコラムに適用したあと、コラム緩衝液のうち0.2M〜0.6M KCl線形塩勾配100〜150mlを用いて湧出させた。湧出させた溶菌物をコラム26X60 S-200 gel filteration(GE healthcare)に適用して高純度の逆転写酵素を得た。コラム分画をSDS-ポリアクリルアミドゲル電気泳動(SDS-PAGE)に適用したあと染色(coomassie brilliant blue staining)を行って分析した。各分画で採取した10μl分画物をそれぞれのゲルレーンで分析した。野生型と類似の分子量(74kDa)のタンパク質を採取し、前記タンパク質の安定性を確保するため、保存液(20mMトリス−塩酸(pH 7.6)、0.1mM EDTA、150mM NaCl、0.1% IGEPAL CA-630(Polyethoxyethanol) 1mM DTT(Dithiothreitol)、50%グリセロール(Glycerol)で透析を進め、突然変異酵素を得て-20℃で保管した。
前記分離した逆転写酵素の活性を確認するため逆転写PCRを行った。逆転写 PCRに用いた物質は次の通りであり、全RNAは5ng/μl〜500、50、5pg/μlの濃度を用いた。
10mM dNTP 2μl
dT(18) 10pmol 1μl
100mM DTT 2μl
RNase抑制剤(50ng/μl) 1μl
蒸留水 7μl
全RNA 2μl
比較例には野生型逆転写酵素を利用し、42℃、50℃、60℃、65℃及び70℃(T306L変異は37℃での反応を追加する)で1時間逆転写PCRを行った。前記で生成されたcDNAを利用してPCRを行った。増幅する遺伝子にはGAPDHを用い(遺伝子の大きさ500bp)、下記塩基配列を有するプライマー対を利用した。
アンチセンス鎖:5'-AGTCCTTCCACGATACCAAAGTTG-3'(配列番号175)
調剤物には下記組成のものを用いた:
PCRプリミックスタイプ(バイオニア)
センスプリマー: 5pmol、1μl
アンチセンスプリマー: 5pmol、1μl
蒸留水 13μl
cDNA 5μl
PCRは95℃ 30秒、57℃ 30秒及び72℃ 30秒の反応条件で30サイクル行い、PCR結果物をアガロースゲルに電気泳動して増幅産物を確認した。
Claims (11)
- 配列番号1に記載されるM-MLV由来の逆転写酵素のアミノ酸配列の63番目のグルタミン(Q63)、264番目のリシン(K264)、295番目のリシン(K295)、306番目のトレオニン(T306)、346番目のグルタミン酸(E346)、408番目のプロリン(P408)、438番目のヒスチジン(H438)及び454番目のアスパラギン(N454)からなる群から選ばれる1つ以上のアミノ酸が他のアミノ酸に置き換えられて熱安定性が増加した逆転写酵素。
- 前記アミノ酸の置換えは、63番目のグルタミンからロイシンへの置換え(Q63L)、264番目のリシンからロイシンへの置換え(K264L)、295番目のリシンからグルタミンへの置換え(K295Q)、306番目のトレオニンからロイシンへの置換え(T306L)、346番目のグルタミン酸からメチオニンへの置換え(E346M)、408番目のプロリンからグルタミン酸への置換え(P408E)、438番目のヒスチジンからチロシンへの置換え(H438Y)、及び454番目のアスパラギンからフェニルアラニンへの置換え(N454F)からなる群から選ばれる1つ以上のアミノ酸置換えを含む請求項1に記載の逆転写酵素。
- 配列番号2ないし配列番号9に記載されるアミノ酸配列からなる群から選ばれるアミノ酸配列を有する請求項2に記載の逆転写酵素。
- 60℃ないし70℃の温度で配列番号1に記載されるアミノ酸配列を有する野生型M-MLV逆転写酵素に比べて優れた逆転写活性を表す請求項1から3の何れかに記載の逆転写酵素。
- 請求項1に記載の熱安定性が増加した逆転写酵素をコーディングする遺伝子。
- 配列番号1に記載されるM-MLV由来の逆転写酵素のアミノ酸配列の63番目のグルタミン(Q63)、264番目のリシン(K264)、295番目のリシン(K295)、306番目のトレオニン(T306)、346番目のグルタミン酸(E346)、408番目のプロリン(P408)、438番目のヒスチジン(H438)及び454番目のアスパラギン(N454)からなる群から選ばれる1つ以上のアミノ酸置換えを含む突然変異逆転写酵素をコーディングする請求項5に記載の遺伝子。
- 配列番号1に記載される野生型逆転写酵素の63番目のグルタミンからロイシンへの置換え(Q63L)、264番目のリシンからロイシンへの置換え(K264L)、295番目のリシンからグルタミンへの置換え(K295Q)、306番目のトレオニンからロイシンへの置換え(T306L)、346番目のグルタミン酸からメチオニンへの置換え(E346M)、408番目のプロリンからグルタミン酸への置換え(P408E)、438番目のヒスチジンからチロシンへの置換え(H438Y)、及び454番目のアスパラギンからフェニルアラニンへの置換え(N454F)からなる群から選ばれる1つ以上のアミノ酸置換えを含む突然変異逆転写酵素をコーディングする請求項6に記載の遺伝子。
- 配列番号11ないし配列番号18に記載される塩基配列からなる群から選ばれる塩基配列を有する請求項7に記載の遺伝子。
- 請求項5に記載の遺伝子を含む発現ベクター。
- 請求項9に記載の発現ベクターに形質転換された形質転換体。
- 請求項1に記載の熱安定性が増加した逆転写酵素を含む逆転写反応用キット。
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