JP2004357634A - Heat-resistant enzyme having sugar nucleotide synthesizing activity and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a sugar nucleotide synthetase having heat-resistance and broad substrate specificity and to stably synthesize a sugar nucleotide useful as a substrate for sugar chain synthesis. <P>SOLUTION: A gene of the sugar nucleotide synthetase has been found from total genom gene information on a super-thermophilic archaebacteria, Sulfolobus tokodaii. The sugar nucleotide synthetase having heat-resistance and broad substrate specificity is produced by a genetic engineering means using the gene and the sugar nucleotide is stably synthesized by using the enzyme. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００１４】
また、この反応においては。上記精製した酵素のみならず、粗酵素であってもよい。例えば、宿主として枯草菌等分泌型の系を用いる場合には、培養液中に本酵素が生成蓄積され、大腸菌等の非分泌型の系を用いる場合には、菌体内に生成されるので、本酵素を含有する培養液あるいはその処理物、もしくは菌体破砕物等の培養処理物を用いて、糖ヌクレオチドを合成してもよい。
以下に、本発明の実施例を示すが、本発明実施例により限定されるものではない。
【００１５】
【実施例１】
糖ヌクレオチド合成酵素の製造
（１）菌の培養
好酸性好気性超好熱古細菌スルフォロバス、トーコーダイイＪＣＭ１０５４５は次の方法で培養した。
１．３ｇの（ＮＨ_４）_２ＳＯ_４、０．２８ｇのＫＨ_２ＰＯ_４、０．２５ｇのＭｇＳＯ_４・７Ｈ_２Ｏ、０．０７ｇのＣａＣｌ_２・２Ｈ_２Ｏ、０．０２ｇのＦｅＣｌ_３・６Ｈ_２Ｏ、１．８ｍｇ のＭｎＣｌ_２・４Ｈ_２Ｏ、４．５ｍｇのＮａ_２Ｂ_４Ｏ_７・１０Ｈ_２Ｏ、０．２２ｍｇのＺｎＳＯ_４・７Ｈ_２Ｏ、０．０５ｍｇのＣｕＣｌ_２・２Ｈ_２Ｏ、０．０３ｍｇのＮａ_２ＭｏＯ_４・２Ｈ_２Ｏ、０．０３ｍｇのＶＯＳＯ_４・ｘＨ_２Ｏ、０．０１ｍｇのＣｏＳＯ_４・７Ｈ_２Ｏ、１．０ｇの酵母エキスを１Ｌの蒸留水に溶かし、この溶液のｐＨを３．５に１０規定Ｈ_２ＳＯ_４溶液で調製した。加圧殺菌した後、ＪＣＭ１０５４５を植菌した。この培養液を８０℃で１〜２日培養し、その後遠心分離し集菌した。
【００１６】
（２）染色体ＤＮＡの調製
ＪＣＭ１０５４５の染色体ＤＮＡは以下の方法により調製した。
培養終了後５０００ｒｐｍ、１０分間の遠心分離により菌体を集菌する。菌体を１０ ｍＭ ＥＤＴＡ（ｐＨ ６．０）溶液で洗浄後、５０ ｍＭ Ｔｒｉｓ／ＨＣｌ−５０ ｍＭ ＥＤＴＡ （ｐＨ８．５）溶液を加えて細胞を溶解させる。さらに、０．５％ Ｎａ−ｌａｕｒｏｙｌｓａｒｃｏｓｉｎａｔｅ、１ ｍｇ／ｍｌ プロテアーゼＫとなるように各々を加えた後、５０℃で３時間保温する。フェノール処理を３回行った後、溶液を１０ ｍＭ Ｔｒｉｓ−１０ ｍＭ ＥＤＴＡ （ｐＨ ８．０）溶液に対して透析する。３７℃で３０分間のＲＮａｓｅによるＲＮＡの分解後、フェノールクロロフォルム溶液で処理した後、１０ ｍＭ Ｔｒｉｓ−１ ｍＭ ＥＤＴＡ（ｐＨ ８．０）で透析を行う。
【００１７】
（３）染色体ＤＮＡを含むショットガンライブラリークローンの作製
実施例２で得られた染色体ＤＮＡを超音波処理することにより断片化した後、アガロースゲル電気泳動により１ｋｂ及び２ｋｂ長のＤＮＡ断片を回収した。この断片をプラスミドベクターｐＵＣ１１８のＨｉｎｃＩＩ制限酵素部位に挿入したショットガンライブラリーを作製した。各ショットガンクローンの末端塩基配列を、ＡＢＩ社製自動塩基配列読み取り装置３７７を用いて解読していった。各ショットガンクローンから得られた塩基配列を塩基配列自動連結ソフトＳｅｑｕｅｎｃｈｅｒを用いて連結編集し、本菌の全塩基配列を決定していった。
【００１８】
（４）ＳＴＲｍｌＡ１−３遺伝子の同定
上記手法で決定された好酸性好気性超好熱古細菌スルフォロバス、トーコーダイイのゲノム塩基配列の大型計算機による解析を行い、グルコース１リン酸チミジル酸結合酵素の機能を含むであろうタンパク質をコードする遺伝子（ＳＴ０４５２， ＳＴ１９７１， ＳＴ２３５２）を３個同定した。この３個の内、超好熱性古細菌スルフォロバストーコーダイイのＳＴ０４５２遺伝子の開始コドンはＡＴＧで、４０１アミノ酸残基のタンパク質をコードする遺伝子として同定された。
【００１９】
（５）発現プラスミドの構築
構造遺伝子領域の前後に制限酵素（ＮｄｅＩとＸｈｏＩ）サイトを構築する目的でＤＮＡプライマーを合成し、ＰＣＲでその遺伝子の前後に制限酵素サイトを導入した。その際に合成されるタンパク質のＣ末端にヒスチジン残基をタグとして結合するように合成されるようにする場合とＳＴ０４５２遺伝子がコードするタンパク質のみを合成させるようにする場合とでプライマーの配列が異なる。
【００２０】
Ｕｐｐｅｒ ｐｒｉｍｅｒ，
５’− ＡＴＡＧＣＡＴＡＴＧＡＡＧＧＣＡＴＴＴＡＴＴＣＴＴＧＣＴＧＣ −３’（配列番号１）
（下線部はＮｄｅＩサイトを示す）
Ｌｏｗｅｒ ｐｒｉｍｅ １， ヒスチジン残基を結合させる場合
５’− ＴＣＡＡＣＴＣＧＡＧＧＡＣＣＴＴＧＡＡＡＡＡＣＴＣＡＣＣ−３’（配列番号２）
（下線部はＸｈｏＩサイトを示す）
Ｌｏｗｅｒ ｐｒｉｍｅ ２， ヒスチジン残基を結合させ無い場合
５’− ＴＣＡＡＣＴＣＧＡＧＣＴＡＧＡＣＣＴＴＧＡＡＡＡＡＣＴＣＡＣＣ −３’（配列番号３）
（下線部はＸｈｏＩサイトを示す）
【００２１】
Ｕｐｐｅｒ ｐｒｉｍｅｒとＬｏｗｅｒ ｐｒｉｍｅｒ１或いはＬｏｗｅｒ ｐｒｉｍｅｒ２を組み合わせたＰＣＲ反応後、制限酵素（ＮｄｅＩとＸｈｏＩ）で完全分解（３７℃で２時間）した後、その構造遺伝子領域断片を精製した。
制限酵素ＮｄｅＩとＸｈｏＩで切断後精製したｐＥＴ２１ｂ（Ｎｏｖａｇｅｎ社製）と上記の構造遺伝子（ＳＴ０４５２）領域断片とをＴ４リガーゼを用いて１６℃、２時間反応させることによって連結した。連結したＤＮＡの一部を大腸菌ＤＨ５αのコンピテントセルに導入し形質転換体のコロニーを得た。得られたコロニーからプラスミドをＱＩＡｐｒｅｐ Ｓｐｉｎ Ｍｉｎｉｐｒｅｐ Ｋｉｔ（ＱＩＡＧＥＮ社製）で精製し、塩基配列を確認して発現プラスミド、ｐＥＴ２１ｂ／ＳＴ０４５２−１及びｐＥＴ２１ｂ／ＳＴ０４５２−２を得た。発現プラスミドｐＥＴ２１ｂ／ＳＴ０４５２−１を用いるとＳＴＲｍｌＡ１はＣ末端にヒスチジンタグが付加された融合タンパク質として生産され、発現プラスミドｐＥＴ２１ｂ／ＳＴ０４５２−２を用いるとＳＴＲｍｌＡ１はＣ末端にヒスチジンタグが付加されないタンパク質として生産される。
【００２２】
（６）組換え遺伝子の発現
大腸菌（Ｅ． ｃｏｌｉ ＢＬ２１（ＤＥ３） ＣｏｄｏｎＰｌｕｓ ＲＩＬ，、Ｎｏｖａｇｅｎ社製）のコンピテントセルを融解して、二本のファルコンチューブに各々０．１ｍｌづつ移す。その中に上記の２種の発現プラスミド１０ｎｇ分に相当する溶液を別々に加え氷中に３０分間放置した後４２℃でヒートショックを３０秒間行い、そこにＳＯＣ培地０．９ｍｌを加え、３７℃で１時間振とう培養する。その後、アンピシリンを含むＬＢ寒天プレート上に適量まき、３７℃で一晩培養し、形質転換体大腸菌 ＢＬ２１（ＤＥ３） ＣｏｄｏｎＰｌｕｓ ＲＩＬ／ｐＥＴ２１ｂ／ＳＴ０４５２−１及び形質転換体大腸菌 ＢＬ２１（ＤＥ３） ＣｏｄｏｎＰｌｕｓ ＲＩＬ／ｐＥＴ２１ｂ／ＳＴ０４５２−２を得た。
【００２３】
当該形質転換体をアンピシリンを含むＬＢ培地（２リットル）中で一晩３７℃において培養した後、ＩＰＴＧ（Ｉｓｏｐｒｏｐｙｌ−ｂ−Ｄ−ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ）を１ｍＭになるように加え、さらに３０℃で５時間培養した。培養後遠心分離（６，０００ｒｐｍ，２０ｍｉｎ）により集菌を行った。
【００２４】
（７）ＳＴＲｍｌＡ１酵素の精製
８リットル培養液から集菌した菌体に２倍量の４０ｍＭトリス塩酸緩衝液（ｐＨ８．０）、１錠のプロテアーゼ阻害剤（Ｃｏｍｐｌｅｔｅ ＥＤＴＡ−ｆｒｅｅ， Ｒｏｃｈｅ社製）、０．５ｍｇのＤｎａｓｅ ＲＱ１（プロメガ社製）を加え懸濁液を得た。得られた懸濁液を超音波破砕し、７５℃で１０分保温した後、遠心分離（１１，０００ ｒｐｍ、２０分）により上清液を得た。この上清液を用いＮｉ−カラム（Ｎｏｖａｇｅｎ， Ｈｉｓ・Ｂｉｎｄ ｍｅｔａｌ ｃｈｅｌａｔｉｏｎ ｒｅｓｉｎ ＆ Ｈｉｓ・Ｂｉｎｄ ｂｕｆｆｅｒ ｋｉｔを使用）による親和性クロマトグラムを行った。ここで得られた０．５ Ｍイミダゾール溶出画分（２０ｍｌ）を、再度７５℃で１０分加熱処理し、遠心分離（１１，０００ ｒｐｍ、２０分）により上清液を得た。次にこの上清液を２０ｍＭトリス塩酸緩衝液（ｐＨ８．０）、２．５Ｍ ＮａＣｌで平衡化したＨｉＴｒａｐ ｐｈｅｎｙｌ ｓｅｐｈａｒｏｓｅ（ファルマシア社製）カラムに吸着させ、同緩衝液中のＮａＣｌ濃度を２．５Ｍから１Ｍに低下させることにより、目的タンパク質の溶出を行った。さらに、セントリプレップＹＭ−５０ （アミコン社）で２ｍｌまで濃縮し、これを２０ｍＭトリス塩酸緩衝液（ｐＨ８．０）、１００ｍＭ ＮａＣｌで透析し、精製サンプルとした。
【００２５】
【実施例２】糖ヌクレオチドの合成
（１）糖ヌクレオチド合成反応（糖とヌクレオチド結合反応）
５０ｍＭ Ｔｒｉｓ緩衝液（ｐＨ７．５）、１２ ｍＭ ＭｇＣｌ_２、２４ ｍＭ Ｇｌｕｃｏｓｅ−１−ｐｈｏｓｐｈａｔｅ、１ ｍＭ ＴＴＰ、１ Ｕのｉｎｏｒｇａｎｉｃ ｐｙｒｏｐｈｏｓｐｈａｔａｓｅからなる酵素反応液３００μｌ中に実施例１で得られた精製酵素０．０１３５ ｍｇを加えた。この酵素反応液を８０℃で保温することにより、反応させた。５分１０分１５分２０分２５分後に３０μｌ を分取し、３００ μｌ の５００ ｍＭ ＫＨ_２ＰＯ_４溶液に加える事により反応停止させた。
【００２６】
（２） 糖ヌクレオチド合成反応（糖とヌクレオチドの結合反応）の測定
ＨＰＬＣを用いて、反応生成物であるＴＤＰ−Ｇｌｕｃｏｓｅの量を、ヌクレオチド部分の紫外線の吸収を目安に測定した。図１に示すように標準物質であるＴＴＰ及びＴＤＰ−Ｇｌｕｃｏｓｅは、ＨＰＬＣにおいて溶出位置が全く異なる。さらに、図２に示すように標準サンプル添加量を変化させたときの、ピークの面積と標準物質量は正確な比例関係にあり、この検量線を用いることにより反応生成物を定量出来る事が示された。
そこで、上記（１）で反応させたサンプルに関しても、ＨＰＬＣで同様の解析を行った。
【００２７】
【実施例３】酵素の性質
（１）タンパク質化学的性質
当該酵素は上記の精製プロセスで完全に精製され、ＳＤＳ−ＰＡＧＥで分子量約４４ＫＤａの単一バンドを示した（図３）。当該酵素は４０１アミノ酸残基より構成され（配列番号４）、そのアミノ酸配列から予測される分子量は４４，０００ Ｄａであった。また、結核菌ＲｍｌＡとの相同性は低いが、ヌクレオチド認識に関与するモチーフが保存されていた（図４）。
【００２８】
（２） 糖ヌクレオチド合成活性（糖とヌクレオチドの結合活性）
当該酵素は３７℃においては、図５に有るように糖ヌクレオチド合成活性をほとんど示さないが、８０℃においては高い酵素活性を示した。活性の発現には一度、８０℃２０分間の加熱処理が必須であるが、加熱後であっても３７℃での活性は非常に低い（図６）。
【００２９】
（３） 熱安定性
５０ｍＭ Ｔｒｉｓ緩衝液（ｐＨ７．５）、１２ ｍＭ ＭｇＣｌ_２、２４ ｍＭ Ｇｌｕｃｏｓｅ−１−ｐｈｏｓｐｈａｔｅ、１ ｍＭ ＴＴＰ、１ Ｕのｉｎｏｒｇａｎｉｃ ｐｙｒｏｐｈｏｓｐｈａｔａｓｅからなる酵素反応液３００μｌ中に、あらかじめ８０℃で５分１０分２０分３０分４０分６０分９０分１２０分間加熱した実施例１で得られた精製酵素０．０１３５ ｍｇを加えた。この酵素反応液を８０℃で５分間保温することにより、反応させた後に３０μｌ を分取し、３００ μｌ の５００ ｍＭ ＫＨ_２ＰＯ_４溶液に加える事により反応停止させた。反応の進行は、実施例２の（２）に有るようにＨＰＬＣで測定した。その結果、図７に示すように、本酵素は８０℃による９０分間の加熱処理後でも、５０％以上の活性を残すことから非常に安定で耐熱性が高いことが示された。
【００３０】
（４）ｐＨ依存性
１２ ｍＭ ＭｇＣｌ_２、２４ ｍＭ Ｇｌｕｃｏｓｅ−１−ｐｈｏｓｐｈａｔｅ、１ ｍＭ ＴＴＰ、１ Ｕのｉｎｏｒｇａｎｉｃ ｐｙｒｏｐｈｏｓｐｈａｔａｓｅからなる酵素反応液３００μｌ中の５０ｍＭ Ｔｒｉｓ緩衝液のｐＨを（ｐＨ ２）、（ｐＨ ４）、（ｐＨ ６）、（ｐＨ ６．５）、（ｐＨ ７）、（ｐＨ ７．５）、（ｐＨ ８）、（ｐＨ １０）に変化させた酵素反応液中に、実施例１で得られた精製酵素０．０１３５ ｍｇを加えた。この酵素反応液を８０℃で５分間保温することにより、反応させた後に３０μｌ を分取し、３００ μｌ の５００ ｍＭ ＫＨ_２ＰＯ_４溶液に加える事により反応停止させた。反応の進行は、実施例２の（２）に有るようにＨＰＬＣで測定した。
図８に示すように、本酵素の活性はｐＨ７．５において最も高い活性を示した。
【００３１】
（５）ヌクレオシド３リン酸基質の多様性
５０ｍＭ Ｔｒｉｓ緩衝液（ｐＨ７．５）、１２ ｍＭ ＭｇＣｌ_２、２４ ｍＭ Ｇｌｕｃｏｓｅ−１−ｐｈｏｓｐｈａｔｅ、１ Ｕのｉｎｏｒｇａｎｉｃ ｐｙｒｏｐｈｏｓｐｈａｔａｓｅ及び１ ｍＭ ｄＴＴＰまたは１ ｍＭ ｄＡＴＰ、１ ｍＭ ｄＧＴＰ、１ ｍＭ ｄＣＴＰ、１ ｍＭ ＵＴＰ、１ ｍＭ ＡＴＰ、１ ｍＭ ＧＴＰ、１ ｍＭ ＣＴＰからなる酵素反応液３００μｌ中に実施例１で得られた精製酵素０．０１３５ ｍｇを加えた。この酵素反応液を８０℃で５分間保温することにより、反応させた後に３０μｌ を分取し、３００ μｌ の５００ ｍＭ ＫＨ_２ＰＯ_４溶液に加える事により反応停止させた。反応の進行は、実施例２の（２）に有るようにＨＰＬＣで測定した。結果を表１に示す。表１から明らかなように、本酵素はｄＴＴＰ以外にｄＡＴＰ、ｄＧＴＰ、ｄＣＴＰ、ＵＴＰを基質として利用出来ることが示された。
【００３２】
【表１】各種ヌクレオシド３リン酸の基質としての利用性

【００３３】
（６）糖１リン酸基質の多様性
５０ｍＭ Ｔｒｉｓ緩衝液（ｐＨ７．５）、１２ ｍＭ ＭｇＣｌ_２、１ ｍＭ ＴＴＰ、１ Ｕのｉｎｏｒｇａｎｉｃ ｐｙｒｏｐｈｏｓｐｈａｔａｓｅ及び２４ ｍＭ α−Ｄ−Ｇｌｕｃｏｓｅ−１−ｐｈｏｓｐｈａｔｅまたは２４ ｍＭ Ｄ−ｆｒｕｃｔｏｓｅ−１−ｐｈｏｓｐｈａｔｅ、２４ ｍＭ α−Ｄ−ｍａｎｎｏｓｅ−１−ｐｈｏｓｐｈａｔｅ、２４ ｍＭ α−Ｄ−Ｇａｌａｃｔｏｓｅ−１−ｐｈｏｓｐｈａｔｅからなる酵素反応液３００μｌ中に実施例１で得られた精製酵素０．０１３５ ｍｇを加えた。この酵素反応液を８０℃で５分間保温することにより、反応させた後に３０μｌ を分取し、３００ μｌ の５００ ｍＭ ＫＨ_２ＰＯ_４溶液に加える事により反応停止させた。反応の進行は、実施例２の（２）に有るようにＨＰＬＣで測定した。表２に示すように、本酵素はα−Ｄ−Ｇｌｕｃｏｓｅ−１−ｐｈｏｓｐｈａｔｅ以外にＤ−ｆｒｕｃｔｏｓｅ−１−ｐｈｏｓｐｈａｔｅ及びα−Ｄ−ｍａｎｎｏｓｅ−１−ｐｈｏｓｐｈａｔｅを基質として利用出来ることが示された。
【００３４】
【表２】各糖一リン酸の基質としての利用性

【００３５】
【発明の効果】
本発明により、試験管内での様々な種類の糖ヌクレオチドを合成することが可能で、かつ熱等に安定な新規な糖ヌクレオチド合成酵素が提供できた。その結果、新規な糖ヌクレオチド合成が可能になった。
一方、糖ヌクレオチドは、糖タンパク質、糖脂質、多糖類の糖鎖合成に糖供与体として機能するものであり、これらの糖鎖合成は、癌転移、器官発生あるいは細胞性免疫等に密接に関連するものとして近年注目されており、本発明は、これら研究の発展において、その貢献度は極めて大きい。
【００３６】
【配列表】

【図面の簡単な説明】
【図１】ＨＰＬＣによる、ＴＴＰ、及びＴＴＰとＴＤＰ−Ｇｌｕｃｏｓｅ混合物の分離パターンを測定したグラフである。
【図２】ＨＰＬＣを用いたＴＤＰ−Ｇｌｕｃｏｓｅの検量線を示す図である。
【図３】精製されたＳＴＲｍｌＡ１蛋白質のＳＤＳ−ＰＡＧＥパターンを示す写真である。
【図４】結核菌（Ｍ．ｔｕｂｅｒｃｕｌｏｓｉｓ）の．ＲｍｌＡ蛋白質と本発明の酵素遺伝子（ＳＴ０４５２，ＳＴ１９７１，ＳＴ２３５２）によりコードされる各蛋白質のアミノ酸配列、及びこれらのタンパク質間で保存された配列モチーフを示す図である。
（なお、数字は、蛋白質アミノ基末端側からの残基数を示す。）
【図５】ＳＴＲｍｌＡ１蛋白質の３７℃における糖ヌクレオチド合成活性を示すグラフである。
【図６】ＳＴＲｍｌＡ１蛋白質の８０℃における糖ヌクレオチド合成活性を示すグラフである。
【図７】ＳＴＲｍｌＡ１蛋白質の８０℃処理における残存活性量を示す図である。
【図８】ＳＴＲｍｌＡ１蛋白質のｐＨの違いによる活性変化を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention uses a heat-resistant protein having a sugar nucleotide synthesizing activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant having the recombinant DNA, and the transformant. The present invention relates to a method for producing a protein having a sugar nucleotide synthesizing activity, and a method for producing a sugar nucleotide using the protein or the transformant.
[0002]
[Prior art]
Examples of enzymes having sugar nucleotide (TDP-Glucose) synthesizing activity include Mycobacterium tuberculosis (see Non-Patent Document 1), Pseudomonas aeruginosa (see Non-Patent Document 2), and Salmonella (Non-Patent Document). The detailed properties of RmlA (Glucose-1-phosphate thymylylyltransferase) derived from Reference 3) have already been reported. RmlA is the first enzyme in the metabolic pathway essential for rhamnose synthesis, and is composed of glucose-1-phosphate and nucleoside. Shi Nucleoside with dotriphosphate as substrate Shi Produces glucose triphosphate. In addition, enzymes that synthesize sugar nucleotides from humans and yeasts have been found, but many of them are derived from normal temperature organisms and are extremely unstable at room temperature or higher, and their activity is rapidly lost by heat treatment at about 80 ° C. Live. For this reason, processing such as sterilization at the time of use is required, and careful storage at a low temperature is required.
[0003]
[Non-Patent Document 1] Yufang Ma, Jonathan A. Mills, John T .; Belisle, Vara Vissa, Mark Howell, Kelly Bowlin, Michael S. Scherman and Michael McNeil "Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase" (1997) Microbiology, 143, 937-945.
[Non-Patent Document 2] Wulf Blankenfeldt, Milliam Asuncion, Joseph S. et al. Lam and James H. Naissmith, "The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA)" (2000) EMBO. , 19, 6652-6663.
[Non-Patent Document 3] Lennart Lindquist, Rudolf Kaiser, Peter R. Reeves and Alf A. Lindberg "Purification, charactrization and HPLC assay of Salmonella glucose-1-phosphate thymidylylyltransferase from the cloned rfb. J. Biochem. , 211, 763-770.
[0004]
[Problems to be solved by the invention]
If a thermostable enzyme having the activity of synthesizing a sugar nucleotide is discovered, it becomes possible to stably synthesize a sugar nucleotide serving as a substrate for sugar chain synthesis. In addition, it is expensive to find an enzyme that can catalyze a binding reaction using various types of sugars required as substrates for sugar chain synthesis as substrates and various types of nucleoside triphosphates such as UTP / GTP as substrates. And synthesis of various unstable sugar nucleotides. In addition, stable enzymes that perform these reactions have been craved.
[0005]
Therefore, an object of the present invention is to provide a novel enzyme having heat resistance and capable of synthesizing sugar nucleotides using various sugars and nucleoside triphosphates as substrates.
[0006]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors have focused on the hyperthermophilic archaeon Sulfolobus tokodaiii strain 7 that grows at 75 to 80 ° C. Searched. Furthermore, an enzyme was produced from the gene using Escherichia coli, and it was confirmed that this enzyme was stably present at high temperature (80 ° C) and showed sugar nucleotide synthesizing activity. The present inventors have found that various kinds of sugar nucleotides can be produced, and have completed the present invention.
[0007]
That is, the present invention relates to the following (1) to (10).
(1) It has the amino acid sequence of SEQ ID NO: 4, 5 or 6, or has one or more amino acid residues deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 4, 5 or 6 A protein having a modified amino acid sequence and a sugar nucleotide synthesizing activity.
(2) DNA encoding the protein of (1).
(3) A DNA having the nucleotide sequence of SEQ ID NO: 7, 8, or 9.
(4) A DNA, which hybridizes with the DNA of SEQ ID NO: 7, 8 or 9 under stringent conditions, and encodes a protein having sugar nucleotide synthesizing activity.
(5) A recombinant DNA, wherein a DNA selected from the DNAs described in (2) to (4) is incorporated into a vector.
(6) A transformant, wherein the recombinant DNA according to (5) has been introduced into a host cell.
(7) A method for producing a protein having a sugar nucleotide synthesizing activity, comprising culturing the transformant according to the above (6) in a medium, and collecting the protein having a sugar nucleotide synthesizing activity from the culture.
(8) A method for producing a sugar nucleotide, comprising allowing the protein according to (1) to act on sugar monophosphate and nucleoside triphosphate.
(9) A method for producing a sugar nucleotide, comprising reacting the culture solution of the transformant or the processed product of the culture according to (6) with sugar monophosphate and nucleoside triphosphate.
(10) A method for producing a sugar nucleotide, comprising allowing a protein encoded by the DNA according to (3) or (4) to act on sugar monophosphate and nucleoside triphosphate.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be specifically described.
The hyperthermophilic archaea used in the present invention is Sulfolobus, an acidophilic aerobic hyperthermophilic archaea, Tokodaii (JCM registration number JCM10545). The three gene regions presumed to exhibit the enzyme activity from the whole genome information of the present hyperthermophilic archaea were amplified and extracted by PCR reaction, inserted into the protein expression plasmid pET21b, and then used for E. coli transformed with the plasmid. This enzyme was produced. The produced enzyme was isolated and purified by heat treatment and column chromatography. Among the purified enzymes, STRmlA1 was found to be a protein having a molecular weight of about 44,000 and an activity of synthesizing various sugar nucleotides.
[0009]
The half-life of this enzyme was 80 ° C. and 50 minutes or more in 50 m Tris-HCl buffer (pH 7.5), indicating high heat resistance.
The amino acid sequence of this STRmlA1 and the base sequence of its gene DNA (ST0452) are shown in SEQ ID NOs: 4 and 7 in the sequence listing, respectively.
The amino acid sequences of the enzymes (STRmlA2 and STRmlA3) encoded by the remaining two gene regions (STRmlA2 and STRmlA3) are shown in SEQ ID NOS: 5 and 6, respectively, and the base sequences of these gene DNAs (ST1971 and ST2352) are shown in SEQ ID NOS: 8 and 9.
[0010]
The enzyme in the present invention is not limited to those having the amino acid sequences shown in SEQ ID NOs: 4 to 6, and in the amino acid sequence, one or more amino acid residues are deleted, substituted, inserted or added. Even so, proteins having this amino acid sequence include those exhibiting sugar nucleotide synthesizing activity. In addition, the enzyme gene DNAs of the present invention are not limited to those having the nucleotide sequences shown in SEQ ID NOs: 7 to 9, but include those encoding the amino acid sequences. Furthermore, DNAs that hybridize to the DNAs represented by any of SEQ ID NOs: 7 to 9 under stringent conditions and encode proteins having sugar nucleotide synthesizing activity are also included. The stringent conditions are as follows: 52.59 g NaCl, 26.46 g sodium citrate, 1 g Ficoll (Type 400), 1 g polyvinylpyrrolidone, 1 g bovine serum albumin, 5 g in 1 liter of hybridization solution SDS, 1 g fragmented salmon sperm DNA, 500 ml formamide, at 42 ° C. Subsequent washing is performed under the conditions that 17.53 g of NaCl, 8.82 g of sodium citrate, and 5 g of SDS are contained in 1 liter of the washing solution, and the temperature is 68 ° C.
[0011]
To obtain the enzyme of the present invention, general genetic engineering techniques can be applied. For example, the above-mentioned various enzyme gene DNAs are inserted into a protein expression plasmid vector such as pET21b, pHY481 or the like to prepare a recombinant vector. A host cell is transformed with the vector, the transformant is cultured in a medium, and the enzyme is purified from the culture, the culture, or the transformant separated and recovered from these cultures by a conventional protein purification means. Purify and isolate. Escherichia coli and Bacillus subtilis can be used as the host cell.
[0012]
In the present invention, a sugar nucleotide is further synthesized using this enzyme. In this synthesis, the enzyme is added to a solution containing a sugar monophosphate and a nucleoside triphosphate, and the reaction temperature is 60 ° C. The reaction is performed at 95 ° C. to obtain a sugar nucleotide.
Examples of the sugar monophosphate include sugar-1-phosphates such as glucose-1-phosphate, mannose-1-phosphate, fructose-1-phosphate and the like, and examples of the nucleoside triphosphate include: TTP (thymidine triphosphate), dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate), GTP (guanosine triphosphate), UTP (uridine triphosphate) And the like.
[0013]
This reaction formula is shown below for the case of synthesizing glucose nucleotides from glucose-1-phosphate and TTP.
Embedded image

[0014]
Also in this reaction. Not only the above purified enzyme but also a crude enzyme may be used. For example, when a secretory system such as Bacillus subtilis is used as a host, the enzyme is produced and accumulated in a culture solution, and when a non-secretory system such as Escherichia coli is used, it is produced in the cells. A sugar nucleotide may be synthesized using a culture solution containing the present enzyme or a processed product thereof, or a cultured product such as a crushed bacterial cell.
Hereinafter, examples of the present invention will be described, but the present invention is not limited to the examples.
[0015]
Embodiment 1
Production of sugar nucleotide synthase
(1) Culture of bacteria
The eosinophilic aerobic hyperthermophilic archaebacterium Sulfolobus, Tokodaii JCM10545 was cultured by the following method.
1.3 g of (NH ₄ ) ₂ SO ₄ 0.28 g KH ₂ PO ₄ 0.25 g of MgSO ₄ ・ 7H ₂ O, 0.07 g CaCl ₂ ・ 2H ₂ O, 0.02 g FeCl ₃ ・ 6H ₂ O, 1.8 mg MnCl ₂ ・ 4H ₂ O, 4.5 mg Na ₂ B ₄ O ₇ ・ 10H ₂ O, 0.22 mg ZnSO ₄ ・ 7H ₂ O, 0.05 mg CuCl ₂ ・ 2H ₂ O, 0.03 mg Na ₂ MoO ₄ ・ 2H ₂ O, 0.03 mg VOSO ₄ ・ XH ₂ O, 0.01 mg CoSO ₄ ・ 7H ₂ O, 1.0 g of yeast extract was dissolved in 1 L of distilled water, and the pH of this solution was adjusted to 3.5 and 10N H. ₂ SO ₄ Prepared in solution. After autoclaving, JCM10545 was inoculated. This culture was cultured at 80 ° C. for 1 to 2 days, and then centrifuged to collect the bacteria.
[0016]
(2) Preparation of chromosomal DNA
Chromosomal DNA of JCM10545 was prepared by the following method.
After completion of the culture, the cells are collected by centrifugation at 5000 rpm for 10 minutes. After washing the cells with a 10 mM EDTA (pH 6.0) solution, a 50 mM Tris / HCl-50 mM EDTA (pH 8.5) solution is added to lyse the cells. Furthermore, after adding each so that it might become 0.5% Na-lauroylsarcosinate and 1 mg / ml protease K, it heat-retains at 50 degreeC for 3 hours. After three phenol treatments, the solution is dialyzed against a 10 mM Tris-10 mM EDTA (pH 8.0) solution. After decomposing RNA by RNase at 37 ° C. for 30 minutes, the cells are treated with a phenol chloroform solution and then dialyzed against 10 mM Tris-1 mM EDTA (pH 8.0).
[0017]
(3) Production of shotgun library clone containing chromosomal DNA
After fragmenting the chromosomal DNA obtained in Example 2 by sonication, 1 kb and 2 kb DNA fragments were recovered by agarose gel electrophoresis. This fragment was inserted into the HincII restriction enzyme site of the plasmid vector pUC118 to prepare a shotgun library. The terminal nucleotide sequence of each shotgun clone was decoded using an automatic nucleotide sequence reader 377 manufactured by ABI. The base sequence obtained from each shotgun clone was linked and edited using base sequence automatic linking software Sequencher, and the entire base sequence of the bacterium was determined.
[0018]
(4) Identification of STRmlA1-3 gene
Analysis of the genomic nucleotide sequence of the acidophilic aerobic hyperthermophilic archaebacterium Sulfolobus and Tokodaii determined by the above method using a large-scale computer, and a gene encoding a protein that would contain the function of glucose 1-phosphate thymidylate synthase (ST0452, ST1971, ST2352) were identified. Of these three, the start codon of the ST0452 gene of the hyperthermophilic archaeon Sulfolobustokodaii was ATG and was identified as a gene encoding a protein of 401 amino acid residues.
[0019]
(5) Construction of expression plasmid
DNA primers were synthesized for the purpose of constructing restriction enzyme (NdeI and XhoI) sites before and after the structural gene region, and restriction enzyme sites were introduced before and after the gene by PCR. The primer sequence is different between the case where synthesis is performed so as to bind a histidine residue as a tag to the C-terminal of the synthesized protein and the case where only the protein encoded by the ST0452 gene is synthesized. .
[0020]
Upper primer,
5'-ATAG CATATG AAGGCATTTATTCTTGCTGC-3 ′ (SEQ ID NO: 1)
(Underlined part indicates NdeI site)
When lower primer 1, histidine residue is bound
5'-TCAA CTCGAG GACCTTGAAAACTCACC-3 ′ (SEQ ID NO: 2)
(The underlined part indicates the XhoI site)
When lower primer 2, histidine residue is not bound
5'-TCAA CTCGAG CTAGACCTTGAAAAAACTCACC-3 '(SEQ ID NO: 3)
(The underlined part indicates the XhoI site)
[0021]
After a PCR reaction combining the Upper primer and Lower primer 1 or Lower primer 2, the fragment was completely digested with restriction enzymes (NdeI and XhoI) (at 37 ° C. for 2 hours), and then the structural gene region fragment was purified.
PET21b (manufactured by Novagen) purified after digestion with restriction enzymes NdeI and XhoI was ligated to the above structural gene (ST0452) region fragment at 16 ° C. for 2 hours using T4 ligase. A part of the ligated DNA was introduced into competent cells of Escherichia coli DH5α to obtain a transformant colony. The plasmid was purified from the obtained colonies using QIAprep Spin Miniprep Kit (manufactured by QIAGEN), and the nucleotide sequence was confirmed to obtain expression plasmids, pET21b / ST0452-1 and pET21b / ST0452-2. When expression plasmid pET21b / ST0452-1 is used, STRmlA1 is produced as a fusion protein with a histidine tag added at the C-terminus, and when expression plasmid pET21b / ST0452-2 is used, STRmlA1 is produced as a protein without a histidine tag at the C-terminus Is done.
[0022]
(6) Expression of recombinant gene
The competent cells of Escherichia coli (E. coli BL21 (DE3) CodonPlus RIL, manufactured by Novagen) are thawed and transferred to two Falcon tubes by 0.1 ml each. The solutions corresponding to 10 ng of the two expression plasmids described above were separately added thereto, left to stand on ice for 30 minutes, heat-shocked at 42 ° C for 30 seconds, and 0.9 ml of SOC medium was added thereto. And shake culture for 1 hour. Thereafter, an appropriate amount was spread on an LB agar plate containing ampicillin, and cultured at 37 ° C. overnight. / ST0452-2 was obtained.
[0023]
After the transformant was cultured overnight at 37 ° C. in an LB medium (2 liters) containing ampicillin, IPTG (isopropyl-b-D-thiogalactopyranoside) was added to 1 mM, and further cultured at 30 ° C. for 5 hours. did. After the culture, the cells were collected by centrifugation (6,000 rpm, 20 min).
[0024]
(7) Purification of STRmlA1 enzyme
The cells collected from the 8 liter culture were added to the cells twice as much as 40 mM Tris-HCl buffer (pH 8.0), 1 tablet of a protease inhibitor (Complete EDTA-free, manufactured by Roche), and 0.5 mg of Dnase RQ1 ( (Promega) was added to obtain a suspension. The resulting suspension was sonicated, kept at 75 ° C. for 10 minutes, and then centrifuged (11,000 rpm, 20 minutes) to obtain a supernatant. Using this supernatant, an affinity chromatogram was performed using a Ni-column (using Novagen, His. Bind metal chelation resin & His. Bind buffer kit). The 0.5 M imidazole elution fraction (20 ml) obtained here was heated again at 75 ° C. for 10 minutes, and centrifuged (11,000 rpm, 20 minutes) to obtain a supernatant. Next, the supernatant was adsorbed on a HiTrap phenyl sepharose (Pharmacia) column equilibrated with 20 mM Tris-HCl buffer (pH 8.0) and 2.5 M NaCl, and the NaCl concentration in the buffer was adjusted to 2.5 M. To 1M to elute the target protein. Furthermore, it was concentrated to 2 ml with Centriprep YM-50 (Amicon), and this was dialyzed against 20 mM Tris-HCl buffer (pH 8.0) and 100 mM NaCl to obtain a purified sample.
[0025]
Example 2 Synthesis of sugar nucleotide
(1) Sugar nucleotide synthesis reaction (sugar and nucleotide binding reaction)
50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 mM TTP, and 1 U of inorganic pyrophosphatase, 300 μl of an enzyme reaction mixture was added with 0.0135 mg of the purified enzyme obtained in Example 1. The enzyme reaction solution was reacted by keeping it warm at 80 ° C. After 5 minutes, 10 minutes, 15 minutes, 20 minutes and 25 minutes, 30 μl was taken and 300 μl of 500 mM KH was removed. ₂ PO ₄ The reaction was stopped by adding to the solution.
[0026]
(2) Measurement of sugar nucleotide synthesis reaction (bonding reaction between sugar and nucleotide)
The amount of the reaction product, TDP-Glucose, was measured using HPLC as a measure of the ultraviolet absorption of the nucleotide portion. As shown in FIG. 1, the elution positions of TTP and TDP-Glucose, which are standard substances, are completely different in HPLC. Further, as shown in FIG. 2, when the amount of the standard sample added was changed, the peak area and the amount of the standard substance were in an accurate proportional relationship, and it was shown that the reaction product could be quantified by using this calibration curve. Was done.
Therefore, the same analysis was performed by HPLC on the sample reacted in the above (1).
[0027]
Example 3 Properties of enzyme
(1) Protein chemical properties
The enzyme was completely purified by the above-described purification process, and showed a single band having a molecular weight of about 44 KDa by SDS-PAGE (FIG. 3). The enzyme was composed of 401 amino acid residues (SEQ ID NO: 4), and the molecular weight predicted from the amino acid sequence was 44,000 Da. In addition, although the homology with R. tuberculosis RmlA was low, the motif involved in nucleotide recognition was preserved (FIG. 4).
[0028]
(2) Sugar nucleotide synthesis activity (sugar-nucleotide binding activity)
The enzyme showed almost no sugar nucleotide synthesis activity at 37 ° C. as shown in FIG. 5, but showed a high enzyme activity at 80 ° C. Heat expression at 80 ° C. for 20 minutes is indispensable once for expression of activity, but the activity at 37 ° C. is very low even after heating (FIG. 6).
[0029]
(3) Thermal stability
50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 mM TTP, 1 U of inorganic pyrophosphatase, in 300 μl of an enzyme reaction solution, preheated at 80 ° C. for 5 minutes 10 minutes 20 minutes 30 minutes 40 minutes 60 minutes 90 minutes 120 minutes 0.0135 mg of the purified enzyme obtained in Example 1 was added. By keeping the enzyme reaction solution at 80 ° C. for 5 minutes, 30 μl was taken after the reaction, and 300 μl of 500 mM KH ₂ PO ₄ The reaction was stopped by adding to the solution. The progress of the reaction was measured by HPLC as in (2) of Example 2. As a result, as shown in FIG. 7, the enzyme remained extremely stable and highly heat-resistant because it retained 50% or more of activity even after heat treatment at 80 ° C. for 90 minutes.
[0030]
(4) pH dependence
12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 mM TTP, and 1 U of inorganic pyrophosphatase. The pH of the 50 mM Tris buffer in 300 μl of the enzyme reaction solution was adjusted to (pH 2), (pH 4), (pH 6), (pH 6). 6.5), (pH 7), (pH 7.5), (pH 8), and (pH 10) in an enzymatic reaction solution, 0.0135 mg of the purified enzyme obtained in Example 1 was added. added. By keeping the enzyme reaction solution at 80 ° C. for 5 minutes, 30 μl was taken after the reaction, and 300 μl of 500 mM KH ₂ PO ₄ The reaction was stopped by adding to the solution. The progress of the reaction was measured by HPLC as in (2) of Example 2.
As shown in FIG. 8, the activity of this enzyme showed the highest activity at pH 7.5.
[0031]
(5) Diversity of nucleoside triphosphate substrates
50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 U of inorganic pyrophosphatase and 1 mM dTTP or 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 1 mM UTP, 1 mM ATP, 1 mM GTP, 1 mM CTP 0.0135 mg of the purified enzyme obtained in Example 1 was added to 300 μl of the liquid. By keeping the enzyme reaction solution at 80 ° C. for 5 minutes, 30 μl was taken after the reaction, and 300 μl of 500 mM KH ₂ PO ₄ The reaction was stopped by adding to the solution. The progress of the reaction was measured by HPLC as in (2) of Example 2. Table 1 shows the results. As is clear from Table 1, it was shown that this enzyme can use dATP, dGTP, dCTP and UTP as substrates in addition to dTTP.
[0032]
Table 1 Availability of various nucleoside triphosphates as substrates

[0033]
(6) Diversity of sugar monophosphate substrates
50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ 1 mM TTP, 1 U of inorganic pyrophosphatase and 24 mM α-D-Glucose-1-phosphate or 24 mM D-frucose-1-phosphate, 24 mM α-D-mannose-1-phosphate, 24 mM α-D. 0.0135 mg of the purified enzyme obtained in Example 1 was added to 300 μl of an enzyme reaction solution containing -Galactose-1-phosphate. By keeping the enzyme reaction solution at 80 ° C. for 5 minutes, 30 μl was taken after the reaction, and 300 μl of 500 mM KH ₂ PO ₄ The reaction was stopped by adding to the solution. The progress of the reaction was measured by HPLC as in (2) of Example 2. As shown in Table 2, it was shown that, in addition to α-D-Glucose-1-phosphate, this enzyme can use D-fractose-1-phosphate and α-D-mannose-1-phosphate as substrates.
[0034]
[Table 2] Availability of each sugar monophosphate as a substrate

[0035]
【The invention's effect】
According to the present invention, a novel sugar nucleotide synthase capable of synthesizing various kinds of sugar nucleotides in a test tube and stable against heat or the like can be provided. As a result, novel sugar nucleotide synthesis has become possible.
On the other hand, sugar nucleotides function as sugar donors in the synthesis of sugar chains of glycoproteins, glycolipids, and polysaccharides, and these sugar chain synthesis is closely related to cancer metastasis, organ development, cellular immunity, etc. In recent years, the present invention has greatly contributed to the development of these studies.
[0036]
[Sequence list]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing a measurement result of a separation pattern of TTP and a mixture of TTP and TDP-Glucose by HPLC.
FIG. 2 is a diagram showing a calibration curve of TDP-Glucose using HPLC.
FIG. 3 is a photograph showing an SDS-PAGE pattern of a purified STRmlA1 protein.
FIG. 4. .M. Tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the amino acid sequence of each protein encoded by RmlA protein and the enzyme gene of this invention (ST0452, ST1971, ST2352), and the sequence motif preserved between these proteins.
(The numbers indicate the number of residues from the amino-terminal side of the protein.)
FIG. 5 is a graph showing sugar nucleotide synthesizing activity of STRmlA1 protein at 37 ° C.
FIG. 6 is a graph showing sugar nucleotide synthesis activity of STRmlA1 protein at 80 ° C.
FIG. 7 is a graph showing the residual activity of STRmlA1 protein after treatment at 80 ° C.
FIG. 8 is a graph showing a change in activity of STRmlA1 protein due to a difference in pH.

Claims (10)

An amino acid sequence having the amino acid sequence of SEQ ID NO: 4, 5 or 6, or having one or more amino acid residues deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 4, 5 or 6 And a sugar nucleotide synthesizing activity. A DNA encoding the protein according to claim 1. A DNA having the nucleotide sequence of SEQ ID NO: 7, 8, or 9. A DNA which hybridizes with the DNA of SEQ ID NO: 7, 8 or 9 under stringent conditions and encodes a protein having sugar nucleotide synthesizing activity. A recombinant DNA, wherein a DNA selected from the DNAs according to claims 2 to 4 is incorporated into a vector. A transformant, wherein the recombinant DNA according to claim 5 has been introduced into a host cell. A method for producing a protein having a sugar nucleotide synthesizing activity, comprising culturing the transformant according to claim 6 in a medium, and collecting a protein having a sugar nucleotide synthesizing activity from the culture. A method for producing a sugar nucleotide, comprising allowing the protein according to claim 1 to act on sugar monophosphate and nucleoside triphosphate. A method for producing a sugar nucleotide, comprising allowing a culture solution of the transformant according to claim 6 or a processed product of the culture to act on sugar monophosphate and nucleoside triphosphate. A method for producing a sugar nucleotide, comprising allowing the protein encoded by the DNA according to claim 3 to act on the sugar monophosphate and the nucleoside triphosphate.

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