JP3819540B2 - Mammalian polypeptide having L-asparaginase activity - Google Patents

Description

【００５４】
表１に示したように、上記のモルモットに由来する野生型ＤＮＡ及びその相同体のうち、ＧＰＡ／ＷＴ ＤＮＡ及びＧＰＡ／Ｄ３６４ｓｔｐ ＤＮＡの発現産物では活性が認められたが、ＧＰＡ／Ｌ３３８ｓｔｐ ＤＮＡ発現産物では活性は検出されなかった。このことは、モルモット由来のＬ−アスパラギナーゼが十分な活性を示すためには、野生型Ｌ−アスパラギナーゼのアミノ酸配列上、第１乃至第３６３のアミノ酸残基よりなる領域があれば十分であることを示唆している。この第１乃至第３６３のアミノ酸残基よりなる配列は、配列表における配列番号４に示したものである。これをコードするＤＮＡの塩基配列は、配列表における配列番号１０に示したものである。またモルモット野生型Ｌ−アスパラギナーゼのアミノ酸配列は、配列表における配列番号５に示されている。
【００５５】
【実験例２−２】
〈ヒト由来のＤＮＡ相同体の発現〉
ヒト由来の野生型ＤＮＡの特定の位置の塩基配列を終止コドン又は他のアミノ酸に対するコドンに置換したＤＮＡ相同体を調製した。先ず、配列表の配列番号１８に示す塩基配列における第１０９６乃至第１０９８の塩基よりなる配列を終止コドンに置換したＤＮＡ相同体を、ＰＣＲ法を適用して調製した。すなわち、鋳型、センスプライマー及びアンチセンスプライマー以外は、全て実験例１−１（ｂ）と同一の条件でＰＣＲを行った。鋳型は、実験例１−２で得たヒト由来のＬ−アスパラギナーゼをコードする野生型ＤＮＡ、センスプライマーは配列が５′−ＡＡＴＣＴＣＧＡＧＣＣＡＣＣＡＴＧＧＣＧＣＧＣＧＣＧＧＴＧ−３′であるオリゴヌクレオチド、アンチセンスプライマーは配列が、５′−ＣＴＧＣＧＧＣＣＧＣＴＣＡＴＴＡＣＡＣＣＧＡＧＧＧＴＧＧＣＧＴ−３′であるオリゴヌクレオチドであった。この結果増幅されたＤＮＡを実験例１−１に従って処理し、組換えＤＮＡ『ｐＣＨＡ／Ｅ３６６ｓｔｐ』を調製し、配列を確認した。ｐＣＨＡ／Ｅ３６６ｓｔｐは、配列表の配列番号１６に併記したアミノ酸配列における第１乃至第３６５のアミノ酸残基よりなる配列をコードするＤＮＡと、その３′末端に介在配列なく存在する終止コドンを含むものであった。以後このコード部分のＤＮＡを『ＨＡ／Ｅ３６６ｓｔｐ ＤＮＡ』と呼ぶ。ＨＡ／Ｅ３６６ｓｔｐＤＮＡは、ＣＭＶプロモーターの下流に５′末端から３′末端方向に連結されていた。
【００５６】
ＤＮＡの特定の位置のコドンを他のアミノ酸に対するコドンへ置換するには、ロバート・エム・ホートンらが『メソッズ・イン・エンザイモロジー』、アカデミック・プレス発行、第２１７巻、２７０乃至２７９頁（１９９３年）で紹介しているオーバーラップ・エクステンション法に従って行った。その概要を図１に示すと共に以下に説明する。第１、変異を導入すべき位置の塩基を、目的とする別の塩基に置換した互いに相補な変異プライマーＡ及び変異プライマーＳを調製する。ここで、変異プライマーＡはアンチセンス鎖であり、変異プライマーＳはセンス鎖である。他方、目的とするＤＮＡの全域を増幅し得るプライマーのセット、すなわち、５′末端プライマー及び３′末端プライマーを調製する。ここで５′末端プライマーはセンス鎖であり、３′末端プライマーはアンチセンス鎖である。第２、基の塩基配列のＤＮＡを鋳型として、先の５′末端プライマーと変異プライマーＡを用いて通常のＰＣＲを行う。これと並行して、同じＤＮＡを鋳型として、先の３′末端プライマーと変異プライマーＳを用いて通常のＰＣＲを行う（第１段ＰＣＲ）。第３、第１段ＰＣＲにより増幅した２つのＤＮＡ、第１段ＰＣＲで使用した５′末端プライマー及び３′末端プライマーを、同一の反応管で混合し、ＰＣＲを行う（第２段ＰＣＲ）。第１段ＰＣＲで増幅された２つのＤＮＡ断片は、鋳型兼プライマーとして変異が導入されたＤＮＡの生成に用いられ、５′末端プライマー及び３′末端プライマーは変異が導入されたＤＮＡ増幅のためのプライマーとして用いられる。この方法により、７とおりの塩基置換を導入したＤＮＡすなわち、７種のＤＮＡ相同体を調製した。７とおりの塩基置換の内容と、それに伴うアミノ酸配列の変化の内容を表２にまとめて示した。この７種のＤＮＡ相同体の調製に用いた、鋳型ＤＮＡと変異プライマーＡ及び変異プライマーＳの配列を表３にまとめて示した。一方、７個のＤＮＡ相同体の調製に用いた、５′末端プライマー及び３′末端プライマーはそれぞれ、この実験例２−２で先に示したｐＣＨＡ／Ｅ３６６ｓｔｐの調製時に用いたセンスプライマー及びアンチセンスプライマーと同一である。
【００５７】
【表２】

【００５８】
【表３】

【００５９】
ここで得たヒト由来のＤＮＡ相同体を実験例１−１に従って処理し、組換えＤＮＡ『ｐＣＨＡ／ＭＵＴ１』、『ｐＣＨＡ／ＭＵＴ２』、『ｐＣＨＡ／ＭＵＴ３』、『ｐＣＨＡ／ＭＵＴ４』、『ｐＣＨＡ／ＭＵＴ５』、『ｐＣＨＡ／ＭＵＴ６』及び『ｐＣＨＡ／ＭＵＴ７』を得た。以後、この実験例２−２で得た、以上のＤＮＡ相同体の発現産物を、『ヒトＬ−アスパラギナーゼ相同体』と呼ぶこともある。同様に配列を確認した後、ＣＯＳ−１細胞へ導入し発現させ、試験した。対照区として、実験例１−２で得たｐＣＨＡ／ＷＴ及びｐＣＤＭ８を同様に処理・試験した。また、この実験例２−２では各発現産物の量的な比較のための参考としてウエスタン・ブロッティングで検出されたバンドのシグナル強度を、デンシトメトリーにより数値化した。以上の結果を表４に示した。
【００６０】
【表４】

【００６１】
表４の結果は、ヒト由来のＬ−アスパラギナーゼは野生型でも、その相同体の一つであるＣ末端欠失変異体（ＨＡ／Ｅ３６６ｓｔｐ ＤＮＡ発現産物）でもモルモット由来のそれらに比べ、比活性が低いことを示唆している。また、これに対し、ヒト由来の野生型Ｌ−アスパラギナーゼ本来のアミノ酸配列の内のいくつかを他のアミノ酸に置換した点突然変異体の中には、検出され得る程度に比活性が上昇するものがあることをも示している。発現産物が少なくとも検出され得る程度の活性を示すことが確認された、ヒト由来のＤＮＡ相同体 ＨＡ／ＭＵＴ１ＤＮＡ、ＨＡ／ＭＵＴ２ ＤＮＡ、ＨＡ／ＭＵＴ３ ＤＮＡ及びＨＡ／ＭＵＴ５ ＤＮＡは、それぞれ配列表における配列番号１１、１２、１３及び１４に示す塩基配列を有すものであり、そのコードするポリペプチドは、それぞれ配列表における配列番号６、７、８及び９に示すアミノ酸配列を有すものである。
【００６２】
以上の実験結果から、哺乳類に由来するポリペプチドが、少なくとも実験例１及び２で用いた発現系・活性測定系で検出され得る程度のＬ−アスパラギナーゼ活性を示すためには、従来公知の、配列表における配列番号１及び２に示すアミノ酸配列の他に、例えば、同じく配列番号３に示すアミノ酸配列を有する必要があることを見出した（ただし、符号「Ｘａａ」を付して示したアミノ酸はグルタミン又はアルギニンを表すものとする）。因みに、モルモット野生型Ｌ−アスパラギナーゼは、そのアミノ酸配列上第２９８乃至第３０２の残基よりなる部分にこの配列を有している。配列表における配列番号１乃至３に示すアミノ酸配列を全て有するポリペプチドとしては、例えば、モルモットに由来する、配列表における配列番号４乃び５に示すアミノ酸配列を有するポリペプチドと、ヒトに由来する配列表における配列番号６乃至９に示すアミノ酸配列を有するポリペプチドが挙げられる。
【００６３】
以上の知見に基づき、本発明者はＬ−アスパラギナーゼ活性を有する哺乳類由来のポリペプチドを発明するに至った。以下実施例に基づきこの発明を説明するが、ここで選択した方法はいずれも斯界において慣用のものである。当然ながら、この発明を実施するための方法は、これらに限定されるわけではない。
【００６４】
【実施例Ａ−１】
〈Ｌ−アスパラギナーゼ活性を有するモルモット由来の野生型ポリペプチド〉
【００６５】
【実施例Ａ−１（ａ）】
〈形質転換体の調製〉
０．５ｍｌ容反応管に１０×ＰＣＲ緩衝液を１０μｌ、２５ｍＭ ｄＮＴＰミックスを１μｌ、鋳型として、実験例１−１で得た組換えＤＮＡ ｐＣＧＰＡ／ＷＴを１ｎｇとり、ＧＰＡ／ＷＴ ＤＮＡの５′末端及び３′末端の配列に基づき化学合成したオリゴヌクレオチドをセンスプライマー又はアンチセンスプライマーとして適量加え、滅菌蒸留水で９９．５μｌとした後、２．５単位／μｌアンプリタックＤＮＡポリメラーゼを０．５μｌ加えた。センスプライマーは、配列が５′−ＧＣＧＡＡＴＴＣＡＴＧＧＣＧＣＧＣＧＣＡＴＣＡ−３′であり、ＧＰＡ／ＷＴ ＤＮＡの５′末端の上流に制限酵素Ｅｃｏ ＲＩ切断部位を付加したものである。アンチセンスプライマーは、配列が５′−ＧＣＡＡＧＣＴＴＴＣＡＧＡＴＧＧＣＡＧＧＣＧＧＣＡＣ−３′であり、ＧＰＡ／ＷＴ ＤＮＡの３′末端の下流に終始コドンを付加し、さらにその下流に制限酵素Ｈｉｎ ｄＩＩＩ切断部位を付加した配列に相補的な配列のものである。常法により、上記混合物を９４℃で１分間、５５℃で１分間、７２℃で３分間の順序でインキュベートするサイクルを４０回繰り返すことによりＰＣＲを行い、ＤＮＡを増幅させた。このＤＮＡを制限酵素Ｅｃｏ ＲＩ及びＨｉｎ ｄＩＩＩで切断することにより約１．７ｋｂｐのＥｃｏ ＲＩ−Ｈｉｎ ｄＩＩＩ断片を得た。このＤＮＡ断片を２５ｎｇとり、これに予め制限酵素Ｅｃｏ ＲＩ及びＨｉｎ ｄＩＩＩで切断しておいたファルマシア製プラスミドベクター『ｐＫＫ２２３−３』を１０ｎｇを加え、さらに宝酒造製ライゲーションキット・バージョン２の溶液Ｉを先のＤＮＡ混合溶液と同体積加えた後、１６℃で２時間インキュベートすることにより、複製可能な組換えＤＮＡ『ｐＫＧＰＡ／ＷＴ』を得た。
【００６６】
組換えＤＮＡ ｐＫＧＰＡ／ＷＴをコンピテントセル法によりファルマシア製大腸菌ＪＭ１０５株に導入し、ここで得られる形質転換体『Ｊ−ＧＰＡ／ＷＴ』を５０μｇ／ｍｌのアンピシリンを含むＬブロス培地（ｐＨ７．２）に接種し、３７℃で１８時間振とう培養した。培養物を遠心分離して形質転換体を採取し、通常のアルカリ−ＳＤＳ法を適用して組換えＤＮＡ ｐＫＧＰＡ／ＷＴを抽出した。蛍光光度計を使用する自動シーケンサで分析することにより、このｐＫＧＰＡ／ＷＴにおいては、図２に示すごとく配列表における配列番号１７に示す塩基配列のＧＰＡ／ＷＴ ＤＮＡがタックプロモーターの下流に５′末端から３′末端方向に連結されているのが確認された。また、ＧＰＡ／ＷＴ ＤＮＡの３′末端側には、介在配列なく終止コドンが存在していることも確認された。
【００６７】
【実施例Ａ−１（ｂ）】
〈ポリペプチドの製造〉
形質転換体Ｊ−ＧＰＡ／ＷＴを５０μｇ／ｍｌのアンピシリンを含むＬブロス培地（ｐＨ７．２）に接種し、３７℃で１８時間振とう培養した。次に３０ｌ容ジャーファーメンタに新鮮なＬブロス培地を１８ｌとり、先に培養しておいた種培養物を１％（ｖ／ｖ）の割合で接種し、３７℃で通気攪拌培養した。培養物の一部を厚さ１ｃｍのキュベットにとり、波長６５０ｎｍにおける吸光度を測定しつつ培養し、吸光度が約１．５に達した時点でＩＰＴＧを終濃度０．１ｍＭとなるように添加し、さらに５時間培養した。その後、遠心分離により培養物から回収される菌体を、１３９ｍＭ塩化ナトリウム、７ｍＭリン酸水素二ナトリウム及び３ｍＭリン酸二水素ナトリウムを含む混液（ｐＨ７．２）に懸濁し常法により超音波処理して菌体を破砕し、菌体破砕物を遠心分離して上清を回収した。
【００６８】
この上清に氷冷下で硫酸アンモニウムを５０％（ｗ／ｖ）まで加え、均一に溶解し、暫時静置し遠心分離後、沈澱を採取した。この沈澱を２０ｍＭトリス塩酸緩衝液（ｐＨ８．０）に溶解させ、同緩衝液に透析後、同緩衝液で平衡化したファルマシア製『キュー・セファロース・エフ・エフ・カラム』に負荷し、同緩衝液で充分に洗浄後、０から０．５Ｍの塩化ナトリウムの濃度勾配下、２０ｍＭトリス塩酸緩衝液（ｐＨ８．０）を通液した。塩化ナトリウム濃度が０．１乃至０．３Ｍ付近で溶出した画分を採取し、膜濃縮しながら１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．５）に溶媒交換した。同緩衝液で平衡化したシグマ製『Ｌ−アスパラギン・アガロース』に負荷し、同緩衝液で洗浄後０．５Ｍ塩化ナトリウムを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．５）で溶出させた。溶出画分を膜濃縮し、１０％（ｖ／ｖ）グリセリンを含むトリス塩酸−塩緩衝液（ｐＨ８．０）で平衡化したファルマシア製『ハイロード・スーパーデックス・２００・カラム』に負荷し、約３００ｋＤａ付近の溶出画分を採取したところ、純度９０％以上の精製ポリペプチドが、培養液あたり約０．１ｍｇ／ｍｌの収量で得られた。
【００６９】
【実施例Ａ−１（ｃ）】
〈理化学的性質〉
精製ポリペプチドを次のように分析し、その理化学的性質を明らかにした。精製ポリペプチドのネイティブな分子量は、実験例１−１（ｅ）に準じてゲル濾過により求めた。その結果、溶出画分のＬ−アスパラギナーゼ活性のピークは分子量約３００ｋＤａに相当する位置に認められた。精製ポリペプチドの、解離した状態での分子量は、実験例１−１（ｅ）中に示したＳＤＳ−ＰＡＧＥにより求めた。その結果、分子量５０±１０ｋＤａの位置に、主たるバンドが認められた。この結果は、精製ポリペプチドは、ネイティブな状態では多量体を形成していることを示している。２種類の方法の測定誤差及び、大腸菌を始めとする哺乳類以外の従来公知のＬ−アスパラギナーゼのネイティブな状態での形態が全て４量体であることを考慮に入れると、この結果は精製ポリペプチドが４量体を形成していることを示していると考えられる。またこの精製ポリペプチドを、実験例１−１（ｄ）に示した方法に供した結果、Ｌ−アスパラギナーゼ活性を有していることが確認された。
【００７０】
【実施例Ａ−２】
〈Ｌ−アスパラギナーゼ活性を有するモルモット由来の野生型ポリペプチド〉
【００７１】
【実施例Ａ−２（ａ）】
〈形質転換体の調製〉
概要を図３に示す。先ず始めに、センスプライマー及びアンチセンスプライマーの配列以外は、全て実施例Ａ−１（ａ）と同一の条件でＰＣＲを行った。センスプライマーの配列は、５′−ＧＴＧＡＡＴＴＣＧＧＡＧＧＴＴＣＡＧＡＴＧＧＣＧＣＧＣＧＣＡＴＣＡ−３′であり、アンチセンスプライマーの配列は、５′−ＣＴＧＣＧＧＣＣＧＣＴＣＡＧＡＴＧＧＣＡＧＧＣＧＧＣＡＣ−３′であった。ここで増幅されたＤＮＡを制限酵素Ｅｃｏ ＲＩ及びＮｏｔ Ｉで切断し、約１．７ｋｂｐのＥｃｏ ＲＩ−Ｎｏｔ Ｉ断片を得た。このＤＮＡ断片７０ｎｇと、予め制限酵素Ｘｈｏ Ｉ及びＮｏｔ Ｉで切断しておいたファルマシア製プラスミドベクター『ｐＢＰＶ』５０ｎｇ及び、リンカーとして次の塩基配列よりなる４種のオリゴヌクレオチドを、それぞれ２５ｎｇずつ混合した。第１のオリゴヌクレオチドの配列は、５′−ＴＣＧＡＧＣＣＡＣＣＡＴＧＡＡＧＴＧＴＴＣＧＴＧＧＧＴＴＡＴＴ−３′、第２のそれは、５′−ＴＴＣＴＴＣＣＴＧＡＴＧＧＣＣＧＴＡＧＴＧＡＣＡＧＧＡＧＴＧ−３′、第３のそれは、５′−ＡＡＴＴＣＡＣＴＣＣＴＧＴＣＡＣＴＡＣＧＧＣＣＡＴＣＡＧＧＡ−３′であり、第４のそれは、５′−ＡＧＡＡＡＡＴＡＡＣＣＣＡＣＧＡＡＣＡＣＴＴＣＡＴＧＧＴＧＧＣ−３′である。なおリンカーとして用いたオリゴヌクレオチドは、いずれも常法に従い合成後、ファルマシア製Ｔ４ポリヌクレオチド・キナーゼを作用させた後、エタノール沈澱により精製したものを用いた。このＤＮＡ混合溶液に、等容の宝酒造製ライゲーション・キット・バージョン２の溶液Ｉを加え、１６℃で２時間保持することにより複製可能な組換えＤＮＡ『ｐＢＩｇＧＰＡ／ＷＴ』を得た。
【００７２】
組換えＤＮＡ ｐＢＩｇＧＰＡ／ＷＴをコンピテントセル法により大腸菌ＨＢ１０１株に導入し、得られる形質転換体を５０μｇ／ｍｌのアンピシリンを含むＬブロス培地（ｐＨ７．２）に接種し、３７℃で１８時間振とう培養した。培養物を遠心分離して菌体を回収し、通常のアルカリ−ＳＤＳ法を適用して組換ＤＮＡ ｐＢＩｇＧＰＡ／ＷＴを抽出した。自動シーケンサで配列を分析すると、このｐＢＩｇＧＰＡ／ＷＴは図４に示すような構造をしていることが確認された。すなわち、モロニー・マウス肉腫ウイルスのロング・ターミナル・リピート由来のエンハンサー（Ｅｍｓｖ）とマウス・メタロチオネインＩ遺伝子由来のプロモーター（Ｐｍｔｉ）からなる転写調節域の下流に、ディー・エフ・スターンが『サイエンス』、第２３５巻、３２１乃至３２４頁（１９８７年）で紹介しているイムノグロブリンの分泌シグナル配列を含むペプチド部分をコードするＤＮＡ、Ｉｇ ｓｅｃ ＤＮＡ が連結されており、さらにその下流に同じ読み枠で、ＧＰＡ／ＷＴ ＤＮＡが５′末端から３′末端方向に連結されていた。また、ＧＰＡ／ＷＴ ＤＮＡの３′末端には介在配列なく終止コドンが存在していることも確認した。
【００７３】
この組換えＤＮＡ ｐＢＩｇＧＰＡ／ＷＴを、ライフ・テクノロジーズ製リポフェクチン試薬を用い、添付のプロトコールに従って、マウス由来細胞株Ｃ１２７（ＡＴＣＣ ＣＲＬ−１６１６）に導入した。該組換えＤＮＡが導入された形質転換体は、第一段階として、増殖の制御能力の喪失すなわちフォーカス形成能によって選択した。第二段階として、フォーカスを含む近傍の細胞を滅菌濾紙にて回収し、目的とする形質転換体を通常の限界希釈法により、Ｌ−アスパラギナーゼ活性の産生能に基づき単細胞化した。斯くして形質転換体『Ｃ−ＧＰＡ／ＷＴ』を得た。
【００７４】
【実施例Ａ−２（ｂ）】
〈ポリペプチドの製造〉
形質転換体Ｃ−ＧＰＡ／ＷＴを、先ず種培養として２．５ｍｌの１０％（ｖ／ｖ）ウシ胎児血清を含むＤＭＥ培地を添加した口径３．５ｃｍのベクトン・ディッキンソン・ラブウェア製６穴マルチウエルプレート『３０４６』の１穴に接種し、コンフルエントにまで培養した。ここから細胞をトリプシン処理することにより剥離させ、その一部を種細胞として、同培地を添加した別の新たな６穴プラスチックプレートの６穴に接種し培養した。同様の操作を順次培養器を拡張させながら繰り返し、細胞数を増加させて、１５０ｃｍ² 培養フラスコ５０本を用いて、該形質転換体を通常の連続培養に供した。最終的に培養上清１００ｌを回収し、実施例Ａ−１（ｂ）の菌体破砕物の上清の処理方法と同様に、硫酸アンモニウム沈澱、沈澱溶解液のキュー・セファロース・エフエフ・カラムを用いたクロマトグラフィー、その溶出画分のＬ−アスパラギン・アガロース・を用いたクロマトグラフィー及びその溶出画分のハイロード・スーパーデックス・２００・カラムを用いたクロマトグラフィーの順で処理した。その結果、純度９０％以上の精製ポリペプチドが、培養液あたり約１μｇ／ｍｌの収量で得られた。
【００７５】
【実施例Ａ−２（ｃ）】
〈理化学的性質〉
得られた精製ポリペプチドの理化学的性質を、実施例Ａ−１（ｃ）と同様に調べると、実施例Ａ−１（ｂ）で得た精製ポリペプチドと同等の性質を示すものであることが確認された。
【００７６】
【実施例Ａ−３】
〈Ｌ−アスパラギナーゼ活性を有するヒト及びモルモット由来のポリペプチド相同体〉
【００７７】
【実施例Ａ−３（ａ）】
〈形質転換体の調製〉
鋳型、センスプライマー及びアンチセンスプライマー以外は実施例Ａ−１（ａ）に示した方法と同一条件でＰＣＲを行い、得られたＤＮＡを実施例Ａ−１（ａ）と同様に処理することにより組換えＤＮＡ『ｐＫＧＰＡ／Ｄ３６４ｓｔｐ』、『ｐＫＨＡ／ＭＵＴ１』、『ｐＫＨＡ／ＭＵＴ２』、『ｐＫＨＡ／ＭＵＴ３』及び『ｐＫＨＡ／ＭＵＴ５』を得た。それぞれの組換えＤＮＡの調製のために用いた鋳型ＤＮＡの名称、センスプライマー及びアンチセンスプライマーの塩基配列を表５にまとめて示す。実施例Ａ−１（ａ）と同様に塩基配列を分析し、これらの組換えＤＮＡの構造を確認した。これらの組換えＤＮＡの構造は、図５乃至９に示している。
【００７８】
【表５】

【００７９】
ここで得た、組換えＤＮＡを同様に実施例Ａ−１（ａ）に従って処理し、形質転換体『Ｊ−ＧＰＡ／Ｄ３６４ｓｔｐ』、『Ｊ−ＨＡ／ＭＵＴ１』、『Ｊ−ＨＡ／ＭＵＴ２』、『Ｊ−ＨＡ／ＭＵＴ３』及び『Ｊ−ＨＡ／ＭＵＴ５』を得た。
【００８０】
【実施例Ａ−３（ｂ）】
〈ポリペプチドの製造〉
これら５種類の形質転換体を実施例Ａ−１（ｂ）と同様に培養、菌体の破砕、菌体破砕物の硫酸アンモニウム沈澱、沈澱溶解液のキュー・セファロース・エフエフ・カラムを用いたクロマトグラフィー、及びその溶出画分のＬ−アスパラギン・アガロースを用いたクロマトグラフィーの順で処理した。この結果得られた溶出画分は実施例Ａ−１（ｂ）と同様に膜濃縮した後、ハイロード・スーパーデックス・２００・カラムを用いたクロマトグラフィーに供し、約１４０ｋＤａ付近の溶出画分を採取したところ、いずれも純度９０％以上の精製ポリペプチドが培養液あたり約０．１ｍｇ／ｍｌの収量で得られた。これらの精製ポリペプチドを実施例Ａ−１（ｃ）の方法により分析し、理化学的性質を明らかにした。実施例Ａ−１で得た結果とあわせて表６に示した。
【００８１】
【表６】

【００８２】
表６に示す結果は、大腸菌を宿主として発現させ精製された、それぞれの野生型ポリペプチド或いはポリペプチド相同体は、いずれもＬ−アスパラギナーゼ活性を示すことが分かる。またこれらのポリペプチドは４量体を形成していることが示されている。
【００８３】
【実施例Ａ−４】
〈Ｌ−アスパラギナーゼ活性を有するヒト及びモルモット由来のポリペプチド相同体〉
【００８４】
【実施例Ａ−４（ａ）】
〈形質転換体の調製〉
鋳型、センスプライマー又はアンチセンスプライマー以外は実施例Ａ−１（ａ）に示した方法と同一条件でＰＣＲを行った。得られたＤＮＡを実施例Ａ−２（ａ）で用いたのと同一のリンカーを用い、同一の条件で連結することにより、組換えＤＮＡ『ｐＢＩｇＧＰＡ／Ｄ３６４ｓｔｐ』、『ｐＢＩｇＨＡ／ＭＵＴ１』、『ｐＢＩｇＨＡ／ＭＵＴ２』、『ｐＢＩｇＨＡ／ＭＵＴ３』及び『ｐＢＩｇＨＡ／ＭＵＴ５』を得た。それぞれの組換えＤＮＡの調製のためのＰＣＲに用いた鋳型ＤＮＡの名称、センスプライマー及びアンチセンスプライマーの塩基配列を表７にまとめて示す。実施例Ａ−１（ａ）と同様に塩基配列を分析し、これらの組換えＤＮＡの構造を確認した。これらの組換えＤＮＡの構造は、図１０乃至１４に示している。
【００８５】
【表７】

【００８６】
ここで得た、組換えＤＮＡを同様に実施例Ａ−２（ａ）と同様に処理することにより、それぞれ形質転換体『Ｃ−ＧＰＡ／Ｄ３６４ｓｔｐ』、『Ｃ−ＨＡ／ＭＵＴ１』、『Ｃ−ＨＡ／ＭＵＴ２』、『Ｃ−ＨＡ／ＭＵＴ３』及び『Ｃ−ＨＡ／ＭＵＴ５』を得た。
【００８７】
【実施例Ａ−４（ｂ）】
〈ポリペプチドの製造〉
【００８８】
これら５種類の形質転換体を実施例Ａ−２（ｂ）と同様に培養し、培養上清を実施例Ａ−１（ｂ）の菌体破砕の処理方法と同様に硫酸アンモニウム沈澱、沈澱溶解液のキュー・セファロース・エフ・エフ・カラムを用いたクロマトグラフィー及びその溶出画分のＬ−アスパラギン・アガロース・ゲルを用いたクロマトグラフィーの順で処理した。この結果得られた溶出画分は実施例Ａ−１（ｂ）と同様に膜濃縮した後、ハイロード・スーパーデックス・２００・カラムを用いたクロマトグラフィーに供し、約１４０ｋＤａ付近の溶出画分を採取したところ、純度９０％以上の精製ポリペプチドが培養液あたり約１μｇ／ｍｌの収量で得られた。これらの精製ポリペプチドを実施例Ａ−１（ｃ）の方法により分析し、理化学的性質を明らかにした。実施例Ａ−３で得た結果とあわせて表８に示した。
【００８９】
【表８】

【００９０】
表８に示す結果は、動物細胞を宿主として発現させ精製された、それぞれの野生型ポリペプチド或いは、ポリペプチド相同体は、いずれもＬ−アスパラギナーゼ活性を示すことが分かる。またこれらのポリペプチドは４量体を形成していることが示される。
【００９１】
以上実施例Ａに示すごとく、この発明のポリペプチドは、いずれもＬ−アスパラギナーゼ活性を示す。したがって、この発明の感受性疾患剤は、ヒトに投与すると、体内のＬ−アスパラギンを加水分解し、Ｌ−アスパラギナーゼ感受性疾患の治療・予防に効果を発揮する。この発明でいう感受性疾患とは、Ｌ−アスパラギン依存性腫瘍細胞の存在に起因する疾患全般を意味するものとし、具体的には、例えば、急性白血病・急性転化した慢性白血病・Ｔ細胞白血病等の白血病、非ホジキン病・ホジキン病等の悪性リンパ腫を挙げることができる。斯くしてこの発明の感受性疾患剤は、上記のごとき感受性疾患を治療・予防するための抗腫瘍剤としての用途を有することとなる。剤型並びに感受性疾患の種類及び性状にもよるが、この発明の感受性疾患剤は、通常、液状、ペースト状又は固状に調製され、当該ポリペプチドを０．０００００１乃至１００％（ｗ／ｗ）、望ましくは、０．０００１乃至１００％（ｗ／ｗ）含んでいる。
【００９２】
この発明の感受性疾患剤は当該ポリペプチド単独の形態はもとより、当該ポリペプチドとそれ以外の生理的に許容される、例えば、基剤、賦形剤、可溶剤、緩衝剤、安定剤、さらには必要に応じて、他の生理活性物質乃至は他の薬剤のうちから選ばれる１種又は２種以上との組成物としての形態をも包含する。基剤、賦形剤、可溶剤、緩衝剤及び安定剤としては具体的には、例えば、日本医薬品添加剤協会編集、『医薬品添加物辞典』（１９９４年）、薬事日報社発行や、日本医薬品添加剤協会編集、『医薬品添加物辞典追補 １９９５』（１９９５年）、薬事日報社発行などに記載のものが挙げられる。他の生理活性物質乃至他の薬剤としては具体的には、例えば、インターフェロン−α、インターフェロン−β、インターフェロン−γ、インターロイキン１、インターロイキン２、インターロイキン３、ＴＮＦ−α、ＴＮＦ−β、ＧＭ−ＣＳＦ、カルボコン、シクロフォスファミド、アクラルビシン、チオテパ、ブスルファン、アンシタビン、シタラビン、フルオロウラシル、テトラヒドロフリルフルオロウラシル、メトトレキサート、アクチノマイシンＤ、クロモマイシンＡ３、ダウノルビシン、ドキソルビシン、ブレオマイシン、メルカプトプリン、プレドニゾロン、マイトマイシンＣ、ビンクリスチン、ビンブラスチン、金コロイド、クレスチン、ピシバニール、レンチナン及び丸山ワクチンなどが挙げられる。
【００９３】
さらに、この発明の感受性疾患剤は、投薬単位形態の薬剤をも包含し、その投薬単位形態の薬剤とは、当該ポリペプチドを、例えば、１回当たりの用量又はその正数倍（４倍まで）若しくはその約数（１／４０まで）に相当する量を含んでおり、投薬に適する物理的に分離した一体の剤型にある薬剤を意味する。この様な投薬形態の薬剤としては、注射剤、液剤、散剤、顆粒剤、錠剤、カプセル剤、舌下剤、点眼剤、点鼻剤、座剤などが挙げられる。
【００９４】
この発明の感受性疾患剤は経口的に投与しても非経口的に投与しても、いずれの場合にも、感受性疾患の治療・予防に効果を発揮する。感受性疾患の種類や症状にもよるが、具体的には、患者の症状や投与後の経過を観察しながら、成人当たり約０．１μｇ乃至５００ｍｇ／回、望ましくは約０．１乃至１００ｍｇ／回のポリペプチドを１乃至４回／日又は１乃至７回／週の用量で１日乃至１年間にわたって経口投与するか、皮内、皮下、筋肉内又は静脈内に非経口投与すればよい。また、この発明の感受性疾患剤のその他の一形態としては、例えば、遺伝子治療を応用した形態が挙げられる。つまり、この発明のポリペプチドをコードするＤＮＡを適宜宿主に導入してなる形質転換体を投与し、この発明のポリペプチドを感受性疾患患者の生体内で産生させることにより、上記の投与形態と同等の効果を発揮する。なお、遺伝子治療を実施するための一般的手順は、例えば、島田隆、斎藤泉、小澤敬也編集、『実験医学別冊バイオマニュアルＵＰシリーズ 遺伝子治療の基礎技術』（１９９６年）、羊土社発行に詳述されている。
【００９５】
次に、実験例３に基づき、この発明のポリペプチドの生物活性について、実験例４に基づき、この発明のポリペプチドの安全性について説明する。
【００９６】
【実験例３】
〈生物活性〉
【００９７】
【実験例３−１】
〈イン・ビトロにおける抗腫瘍細胞効果〉
【００９８】
ヒト組織球リンパ腫細胞株Ｕ９３７（ＡＴＣＣ Ｎｏ．ＣＲＬ−１５９３）及びヒトリンパ芽球由来細胞株Ｍｏｌｔ４（ＡＴＣＣ Ｎｏ．ＣＲＬ−１５８２）を、１０％（ｖ／ｖ）ウシ胎児血清を含むＲＰＭＩ１６４０培地で予め継代培養しておいた。対数増殖期にあるそれぞれの細胞培養系から、細胞を遠心分離により回収し、同培地で２×１０^５個／ｍｌの細胞濃度に調整し、該細胞懸濁液をベクトン・ディッキンソン・ラブウェア製２４穴マルチウエルプレート『３０４７』プレートに、１穴あたり１ｍｌで合計１３穴に接種した。ここに実施例Ａ−１乃至Ａ−４で調製した１２種類の精製ポリペプチドのＰＢＳによる希釈液をそれぞれ添加し、５％（ｖ／ｖ）ＣＯ_２インキュベーター内で、３７℃で７２時間培養した。各精製ポリペプチドの終濃度は、Ｌ−アスパラギナーゼ活性として１単位／ｍｌであった。対照区として、ＰＢＳを等容添加して同じく培養する系を設けた。培養後細胞を回収し、トリパン・ブルーにより死細胞を染色して、精製ポリペプチド添加系の細胞生存率を対照区と比較した。その結果、いずれの精製ポリペプチドを添加した系も、細胞生存率は対照区と比較して有意に低い値を示した。このことは、実施例Ａ−１乃至Ａ−４で得られた精製ポリペプチドは、いずれもＵ９３７及びＭｏｌｔ４に対する細胞障害性を有することを示している。
【００９９】
【実験例３−２】
〈イン・ビボにおける抗腫瘍細胞効果〉
【０１００】
東北大学加齢医学研究所医用細胞資源センターに登録されているマウスリンパ腫細胞株６Ｃ３ＨＥＤを、常法により１×１０^７個／匹で８日ごとにその脇腹に皮下注射することによって継代移植されているＣ３Ｈマウスをモデルマウスとして用いた。該細胞の移植後４日目から７日目までの毎日、該モデルマウスに実施例Ａ−１乃至Ａ−４で得られた精製ポリペプチドを、４００単位／匹で、静脈注射にて投与した。移植後４及び８日目の腫瘍の大きさを肉眼で観察した。なお精製ポリペプチドは、０．１５Ｍの塩化ナトリウム溶液で希釈した後、ミリポア製の孔径０．４５μｍのメンブランフィルターで濾過した後に投与した。対照区として、０．１５Ｍ塩化ナトリウム溶液を同様に処理した系を設けた。その結果、対照区では腫瘍の明らかな肥大が認められたのに対し、精製ポリペプチドを投与した系では腫瘍の明らかな退縮又は消失が認められた。このことは、実施例Ａ−１乃至Ａ−４で調製される精製ポリペプチドが、いずれもモデルマウスの腫瘍を治癒する能力があることを示している。
【０１０１】
【実験例４】
〈急性毒性試験〉
実施例Ａ−１乃至Ａ−４で調製された精製ポリペプチドをそれぞれ別個に、常法にしたがって８週齢のマウスに経皮、経口あるいは腹腔内に注射投与した。その結果、これらの精製ポリペプチドのＬＤ５０は、いずれの投与経路による場合も約１００ｍｇ／ｋｇ以上であった。このことは、この発明のポリペプチドがヒトへの投与を前提とする医薬品として安全であることを裏付けている。
【０１０２】
以下、この発明の感受性疾患剤につき実施例を挙げて説明する。
【０１０３】
【実施例Ｂ】
〈感受性疾患剤〉
【０１０４】
【実施例Ｂ−１】
〈液剤〉
安定剤として１％（ｗ／ｖ）のヒト血清アルブミンを含む生理食塩水に実施例Ａ−１乃至Ａ−４で得た精製ポリペプチドをそれぞれ別個に０．１ｍｇ／ｍｌとなるように溶解させ、常法に従った精密濾過により滅菌して液剤を得た。
【０１０５】
いずれの製品も安定性に優れ、悪性腫瘍、急性白血病、悪性リンパ腫、慢性白血病の急性転化、Ｔ細胞白血病を含む感受性疾患を治療・予防するための注射剤、点眼剤、及び点鼻剤として有用である。
【０１０６】
【実施例Ｂ−２】
〈液剤〉
安定剤として１％（ｗ／ｖ）のグリセリンを含む生理食塩水に実施例Ａ−１乃至Ａ−４で得た精製ポリペプチドをそれぞれ別個に０．１ｍｇ／ｍｌとなるように溶解させ、常法に従った精密濾過により滅菌して液剤を得た。
【０１０７】
いずれの製品も安定性に優れた、悪性腫瘍、急性白血病、悪性リンパ腫、慢性白血病の急性転化、Ｔ細胞白血病を含む感受性疾患を治療・予防するための注射剤、点眼剤、及び点鼻剤として有用である。
【０１０８】
【実施例Ｂ−３】
〈乾燥注射剤〉
安定剤として、１％（ｗ／ｖ）の精製ゼラチンを含む生理食塩水に実施例Ａ−１乃至Ａ−４で得た精製ポリペプチドをそれぞれ別個に５０ｍｇ／ｍｌとなるように溶解させ、常法に従った精密濾過により滅菌して、バイアル瓶に１ｍｌずつ分注し、凍結乾燥後密栓した。
【０１０９】
いずれの製品も安定性に優れ、悪性腫瘍、急性白血病、悪性リンパ腫、慢性白血病の急性転化、Ｔ細胞白血病を含む感受性疾患を治療・予防するための乾燥注射剤として有用である。
【０１１０】
【実施例Ｂ−４】
〈軟膏剤〉
滅菌蒸留水に和光純薬工業製カルボキシビニルポリマー『ハイビスワコー１０４』と高純度トレハロースをそれぞれ濃度１．４％（ｗ／ｗ）及び２．０％（ｗ／ｗ）になるように溶解させ、実施例Ａ−１乃至Ａ−４で得た精製ポリペプチドをそれぞれ別個に均一に混合後、ｐＨ７．２に調整して１ｇ当たり該ポリペプチドを約１０ｍｇ含むペースト状物を得た。
【０１１１】
いずれの製品も延展性と安定性に優れ、悪性腫瘍、急性白血病、悪性リンパ腫、慢性白血病の急性転化、Ｔ細胞白血病を含む感受性疾患を治療・予防するための軟膏として有用である。
【０１１２】
【実施例Ｂ−５】
〈錠剤〉
林原製無水結晶α−マルトース粉末『ファイントース』に実施例Ａ−１乃至Ａ−４で得た精製ポリペプチドと細胞賦活剤としてのルミンを均一に混合し、得られる混合物を打錠機により打錠して製品１錠（約２００ｍｇ）当たり該ポリペプチド及びルミンをそれぞれ約５ｍｇ含む錠剤を得た。
【０１１３】
いずれの製品も摂取性と安定性に優れ、さらに細胞賦活作用も有し、悪性腫瘍、急性白血病、悪性リンパ腫、慢性白血病の急性転化、Ｔ細胞白血病を含む感受性疾患を治療・予防するための錠剤として有用である。
【０１１４】
【発明の効果】
この発明は、Ｌ−アスパラギナーゼ活性を有する哺乳類由来のポリペプチドの発見に基づくものである。この発明のポリペプチドは、いずれもアミノ酸配列まで解明されている物質であり、安定したＬ−アスパラギンを加水分解する活性を有する。これにより、この発明のポリペプチドは、Ｌ−アスパラギン依存性腫瘍細胞に起因する各種の疾患に対する治療剤・予防剤として威力を発揮する。
【０１１５】
この発明のポリペプチドは哺乳類由来であることから、ヒトに対する抗原性が低く、多量投与或いは継続投与しても重篤な副作用を惹起することがない。したがって、この発明のポリペプチドは、使用に際して患者の感受性に関して厳密な管理をしなくとも、所望の効果を発揮できる利点がある。
【０１１６】
かくも有用なるこの発明のポリペプチドは、これをコードするこの発明のＤＮＡを利用することにより、所望量を容易に製造することができる。
【０１１７】
この発明は、斯くも顕著な作用効果を発揮するものであり、斯界に貢献すること誠に多大な意義のある発明であるといえる。
【０１１８】
【配列表】

【０１１９】

【０１２０】

【０１２１】

【０１２２】

【０１２３】

【０１２４】

【０１２５】

【０１２６】

【０１２７】

【０１２８】

【０１２９】

【０１３０】

【０１３１】

【０１３２】

【０１３３】

【０１３４】

【０１３５】

【図面の簡単な説明】
【図１】オーバー・ラップ・エクステンション法の概要を示す図である。
【図２】組換えＤＮＡ ｐＫＧＰＡ／ＷＴの制限酵素地図を示す図である。
【図３】組換えＤＮＡ ｐＢＩｇＧＰＡ／ＷＴの調製法の概要を示す図である。
【図４】組換えＤＮＡ ｐＢＩｇＧＰＡ／ＷＴの制限酵素地図を示す図である。
【図５】組換えＤＮＡ ｐＫＧＰＡ／Ｄ３６４ｓｔｐの制限酵素地図を示す図である。
【図６】組換えＤＮＡ ｐＫＨＡ／ＭＵＴ１の制限酵素地図を示す図である。
【図７】組換えＤＮＡ ｐＫＨＡ／ＭＵＴ２の制限酵素地図を示す図である。
【図８】組換えＤＮＡ ｐＫＨＡ／ＭＵＴ３の制限酵素地図を示す図である。
【図９】組換えＤＮＡ ｐＫＨＡ／ＭＵＴ５の制限酵素地図を示す図である。
【図１０】組換えＤＮＡ ｐＢＩｇＧＰＡ／Ｄ３６４ｓｔｐの制限酵素地図を示す図である。
【図１１】組換えＤＮＡ ｐＢＩｇＨＡ／ＭＵＴ１の制限酵素地図を示す図である。
【図１２】組換えＤＮＡ ｐＢＩｇＨＡ／ＭＵＴ２の制限酵素地図を示す図である。
【図１３】組換えＤＮＡ ｐＢＩｇＨＡ／ＭＵＴ３の制限酵素地図を示す図である。
【図１４】組換えＤＮＡ ｐＢＩｇＨＡ／ＭＵＴ５の制限酵素地図を示す図である。
【符号の説明】
Ｅｃｏ ＲＩ Ｅｃｏ ＲＩ切断部位
Ｈｉｎ ｄＩＩＩ Ｈｉｎ ｄＩＩＩ切断部位
Ｎｏｔ Ｉ Ｎｏｔ Ｉ切断部位
Ｘｈｏ Ｉ Ｘｈｏ Ｉ切断部位
ＧＰＡ／ＷＴ モルモット野生型Ｌ−アスパラギナーゼをコードするＤＮＡ（ＧＰＡ／ＷＴ ＤＮＡ）
Ｄ３６４ｓｔｐ モルモットＬ−アスパラギナーゼ相同体をコードするＤＮＡ（ＧＰＡ／Ｄ３６４ｓｔｐＤＮＡ）
ＨＡ／ＭＵＴ１ ヒトＬ−アスパラギナーゼ相同体をコードするＤＮＡ（ＨＡ／ＭＵＴ１ ＤＮＡ）
ＨＡ／ＭＵＴ２ ヒトＬ−アスパラギナーゼ相同体をコードするＤＮＡ（ＨＡ／ＭＵＴ２ ＤＮＡ）
ＨＡ／ＭＵＴ３ ヒトＬ−アスパラギナーゼ相同体をコードするＤＮＡ（ＨＡ／ＭＵＴ３ ＤＮＡ）
ＨＡ／ＭＵＴ５ ヒトＬ−アスパラギナーゼ相同体をコードするＤＮＡ（ＨＡ／ＭＵＴ５ ＤＮＡ）
Ｐｔａｃ ｔａｃプロモーター
ｒｒｎＢＴ１Ｔ２ リボゾームＲＮＡオペロンの転写終領域
５Ｓ ５ＳリボゾームＲＮＡ遺伝子
ＡｍｐＲ アンピシリン耐性遺伝子
ｐＢＲ３２２ｏｒｉ 大腸菌における複製開始点
Ｉｇ ｓｅｃ イムノグロブリンの分泌シグナル配列を含むペプチド部分をコードするＤＮＡ（Ｉｇ ｓｅｃ ＤＮＡ）
Ｅｍｓｖ モロニー・マウス肉腫ウイルスのロング・ターミナル・リピート由来のエンハンサー
Ｐｍｔｉ マウス・メタロチオネインＩ遺伝子由来のプロモーター
Ｐｏｌｙ（Ａ） ＳＶ４０由来のポリ（Ａ）付加シグナル
ＢＰＶＩ ウシパピローマ・ウイルスのゲノム[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel polypeptide having L-asparaginase activity, and particularly to a polypeptide derived from a mammal having L-asparaginase activity.
[0002]
[Prior art]
L-asparaginase (EC 3.5.1.1) is an enzyme that catalyzes a reaction that hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. The study of L-asparaginase as an anti-tumor agent was carried out by J. G. Kids in the Journal of Experimental Medicine, Vol. 98, pages 565-582 (1953) against guinea pig serum against lymphoma. It originates in reporting that anti-tumor action is recognized. Later, JD Bloom revealed in Nature, Vol. 191, pp. 1114 to 1115 (1961) that the substance of this antitumor activity was L-asparaginase. Currently, the mechanism of action is explained as follows. That is, since tumor cells such as acute lymphocytic leukemia cells lack L-asparagine synthetase, L-asparagine in the living body becomes an essential nutrient, and therefore L-asparagine is deficient or does not exist in the living body. You can't grow under the conditions, so you can't stay alive. However, since L-asparagine is not an essential nutrient for normal cells, in a malignant tumor patient, when L-asparagine is hydrolyzed in vivo by L-asparaginase, only the tumor cells are selectively killed, and the malignant tumor is removed. It can be treated. Since then, energetic research has continued with the aim of putting L-asparaginase into practical use as an antitumor agent. As a result, L-asparaginase derived from Escherichia coli has now been used as a therapeutic agent for leukemia and lymphoma. .
[0003]
However, L-asparaginase derived from Escherichia coli is a heterogeneous protein, as seen from the human body, and conventional therapeutic agents that use this protein are anaphylactic shock, urticaria, edema, wheezing, dyspnea when administered to patients. Serious side effects such as hypersensitivity reactions such as Thus, conventional therapeutic agents are in a situation in which the dose and administration frequency must be greatly limited, and therefore several proposals have been made to reduce or eliminate such side effects.
[0004]
First, as seen in JP-A No. 54-119082, 65% or more of amino groups in L-asparaginase derived from Escherichia coli by 2-O-substituted polyethylene glycol-4,6-dichloro-S-triazine. In order to chemically modify the L-asparaginase itself. The second is to collect human L-asparaginase from the culture or human urine of certain human cell lines, as seen in Japanese Patent Application Laid-Open No. 4-320684 or Japanese Patent Application Laid-Open No. 55-19018. It is. The first proposal has the advantage that L-asparaginase of Escherichia coli, which is easily available in large quantities, can be used, but the problem is that the modification reaction is difficult to control and the side effects are not completely eliminated. There is. The human L-asparaginase according to the second proposal, unlike that produced by Escherichia coli, has the advantage that it is difficult to produce antibodies even when administered to a patient. However, human cells disclosed in JP-A-4-320684 are disclosed. Has a problem that L-asparaginase production ability is not sufficiently high, and there is a problem that a large amount of cells must be cultured when trying to mass-produce L-asparaginase, and the method disclosed in JP-A-55-19018 However, there is a problem that it is difficult to obtain fresh human urine continuously on an industrial scale.
[0005]
By the way, recent advances in recombinant DNA technology are remarkable. Today, if DNA encoding the polypeptide of interest can be isolated, a recombinant DNA is produced from the DNA and a vector capable of autonomous replication, and this is introduced into cells of microorganisms or animals and plants. By culturing the transformant, a desired amount of the polypeptide can be easily obtained. To date, however, mammalian DNA encoding L-asparaginase has not yet been isolated,
Of course, mammalian L-asparaginase has not been produced by recombinant DNA technology.
[0006]
In view of such a situation, in this field, DNA derived from a mammal encoding an active L-asparaginase is isolated as soon as possible, and by applying recombinant DNA technology to the isolated DNA, L-asparaginase Establishment of a technique for mass-producing a polypeptide derived from a mammal having activity is desired.
[0007]
[Problems to be solved by the invention]
A first object of the present invention is to provide a polypeptide derived from a mammal having L-asparaginase activity.
[0008]
A second object of the present invention is to provide a DNA encoding such a polypeptide.
[0009]
A third object of the present invention is to provide a recombinant DNA comprising a DNA encoding such a polypeptide and a vector capable of autonomous replication.
[0010]
A fourth object of the present invention is to provide a transformant obtained by appropriately introducing such a recombinant DNA into a host.
[0011]
A fifth object of the present invention is to provide a method for producing the above polypeptide using such a transformant.
[0012]
The sixth object of the present invention is to provide a sensitive disease agent comprising such a polypeptide as an active ingredient.
[0013]
[Means for Solving the Problems]
This invention solves said 1st subject with polypeptide derived from the mammal which has L-asparaginase activity.
[0014]
In addition, the present invention solves the second problem by using a DNA encoding a mammal-derived polypeptide having L-asparaginase activity.
[0015]
In addition, the present invention solves the third problem by a recombinant DNA comprising a DNA encoding a mammalian polypeptide having L-asparaginase activity and a vector capable of autonomous replication.
[0016]
In addition, the present invention solves the fourth problem by a transformant obtained by appropriately introducing a DNA encoding a mammalian polypeptide having L-asparaginase activity into a host.
[0017]
The present invention also provides the fifth problem by cultivating a transformant obtained by appropriately introducing a DNA encoding a mammal-derived polypeptide having L-asparaginase activity into a host, and removing the produced polypeptide from the culture. The problem is solved by the method for producing the collected polypeptide.
[0018]
The present invention also solves the sixth problem by a sensitive disease agent comprising, as an active ingredient, a mammal-derived polypeptide having L-asparaginase activity.
[0019]
[Action]
The polypeptide of the present invention derived from a mammal acts on L-asparagine to produce L-aspartic acid and ammonia.
[0020]
The DNA of this invention is inserted into an appropriate vector capable of autonomous replication to form a recombinant DNA, and this recombinant DNA is usually introduced into a suitable host that does not produce the polypeptide but can be easily propagated. By producing a transformant, the production of the polypeptide is expressed.
[0021]
The replicable recombinant DNA of this invention does not usually produce the polypeptide, but expresses the production of the polypeptide by introducing it into a suitable host that can be easily propagated to form a transformant. .
[0022]
The transformant of this invention expresses production of the polypeptide when cultured.
[0023]
If this transformant is cultured according to the production method of the present invention, a desired amount of polypeptide can be easily obtained.
[0024]
The sensitive disease agent of the present invention exhibits a remarkable therapeutic / preventive effect without serious side effects when administered to humans.
[0025]
The inventor of the present invention has isolated the guinea pig and human-derived DNA encoding L-asparaginase for the first time in the world and succeeded in elucidating the base sequence. That is, it was clarified that the guinea pig-derived DNA has the base sequence shown in SEQ ID NO: 15 in the sequence listing, and the human-derived DNA has the base sequence shown in SEQ ID NO: 16 in the sequence listing. This finding is attributed to the same applicant. Special Kaihei 8-214885 public In the news It is disclosed. The present invention is based on these findings and provides for the first time in the world a polypeptide derived from a mammal having L-asparaginase activity.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
The polypeptide of this invention does not ask | require the origin and origin as long as it is derived from mammals and has L-asparaginase activity. The polypeptide of the present invention can be obtained by expressing a gene derived from a mammal, and usually contains the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3 in the sequence listing. However, in the amino acid sequence shown in SEQ ID NO: 3, the amino acid indicated by the symbol “Xaa” represents glutamine or arginine. Examples of the individual polypeptides include polypeptides having any one of the amino acid sequences shown in SEQ ID NOs: 4 to 9 in the sequence listing. However, in view of the state of the art, with respect to the amino acid sequences shown in SEQ ID NOs: 4 to 9, one or more of the amino acids are replaced with other amino acids without substantially losing L-asparaginase activity. It is relatively easy to replace. On the other hand, even if the polypeptides are derived from the same DNA, the type of vector used for the introduction thereof, the type of host to which the polypeptide is introduced, or the components of the medium used for culturing the transformant containing the DNA Depending on the composition, culture temperature, pH, etc., one or two amino acids at the N-terminus and / or C-terminus are retained in the process of modification and purification after DNA expression by the host enzyme, although the desired activity is retained. One or more amino acids may be deleted, or one or more amino acids may be added to the N-terminal and / or C-terminal, or a sugar chain may be added to the produced polypeptide. In view of such a situation, it goes without saying that the polypeptide has any amino acid sequence shown in SEQ ID NO: 4 to 9 in the sequence listing as it is, and as long as it has L-asparaginase activity, its homologues are naturally also present in the present invention. Is included. The polypeptide of the present invention usually has L-asparaginase activity when in the form of a multimer, preferably in the form of a tetramer.
[0027]
The polypeptides of this invention are usually produced by recombinant DNA technology. That is, the polypeptide of the present invention can be usually produced by culturing a transformant containing DNA encoding it and collecting the produced polypeptide from the culture. For example, such a transformant appropriately converts a recombinant DNA containing any one of the nucleotide sequences shown in SEQ ID NOs: 10 to 15 in the sequence listing. Introduce to host Can be obtained. In the above base sequence, one or two or more bases may be replaced with another base without changing the encoded amino acid sequence by utilizing the degeneracy of the gene code. In addition, in order to promote production of the polypeptide in a DNA host, one or more bases in the base sequence encoding the polypeptide or a homologue thereof can be appropriately substituted with other bases. Needless to say. Furthermore, a sequence encoding one or more amino acids and / or a sequence not encoding an amino acid is added to the 5 ′ end and / or 3 ′ end of the base sequence encoding the polypeptide or a homologue thereof. Needless to say, it can be done.
[0028]
The DNA encoding the polypeptide of the present invention is artificially synthesized even if it is obtained from nature as long as the polypeptide as its expression product has L-asparaginase activity. It may be a wild-type DNA whose sequence matches that obtained from nature, or a DNA homologue to the wild-type DNA. Examples of the natural source of DNA encoding the polypeptide of the present invention include guinea pig liver, from which wild-type DNA containing the nucleotide sequence shown in SEQ ID NO: 15 is obtained by a conventional method. It is done. That is, by the same applicant Special Kaihei 8-214885 public In the news As disclosed, first, a cDNA library is prepared by a conventional method using poly (A) -added RNA purified from guinea pig liver as a material. Here, a plaque hybridization method was applied using an oligonucleotide chemically synthesized based on a partial amino acid sequence of L-asparaginase purified from guinea pig serum as a probe, and a phage clone containing a DNA encoding the polypeptide of the present invention was obtained. Collect. If the phage clones thus obtained are usually treated by a general method, the DNA can be obtained. It is also possible to chemically synthesize DNA based on SEQ ID NO: 15 in the sequence listing. Examples of homologues to wild-type DNA include, for example, individual DNAs containing the base sequences shown in SEQ ID NOs: 10 to 14 in the sequence listing. DNA containing the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing is converted to wild-type DNA obtained as described above shown in SEQ ID NO: 15 in the sequence listing by methods commonly used in the art such as PCR and point mutation. The introduction method can be obtained by applying based on the base sequence of SEQ ID NO: 10 in the sequence listing. Any of the DNAs containing the base sequences shown in SEQ ID NOs: 11 to 14 in the sequence listing can be obtained as follows. That is, first, by the same applicant Special Kaihei 8-214885 public In the news By screening a human liver cDNA library or the like as disclosed, wild-type DNA containing the base sequence shown in SEQ ID NO: 16 in the sequence listing is obtained. Then, a PCR method or a point mutation introduction method commonly used in this field may be applied to the wild-type DNA based on the nucleotide sequences of SEQ ID NOs: 11 to 14 in the sequence listing. It is also possible to chemically synthesize DNA based on the base sequences shown in SEQ ID NOs: 10 to 14 in the sequence listing.
[0029]
Such DNA is usually introduced into the host in the form of recombinant DNA. Recombinant DNA usually comprises a vector that can autonomously replicate with DNA, and if DNA is available, it can usually be prepared relatively easily by general recombinant DNA techniques. Examples of such vectors include plasmid vectors such as pKK223-3, pGEX-2T, pRL-λ, pBTrp2 DNA, pUB110, YEp13, Ti plasmid, Ri plasmid, pBI121, pCDM8, pBPV, BCMGSneo, Of these, pKK223-3, pGEX-2T, pRL-λ, pBTrp2 DNA and pUB110 are used for expression of the DNA of the present invention in prokaryotic cells such as Escherichia coli and Bacillus subtilis. YEp13, Ti plasmid, Ri plasmid, pBI121, pCDM8, pBPV and BCMGSneo are preferred for expression in cells.
[0030]
In order to insert the DNA of the present invention into such a vector, a general method is usually employed in this field. Specifically, for example, an autonomously replicable vector is first cleaved with a restriction enzyme. Next, the PCR method is applied to introduce the same restriction enzyme cleavage sites as those previously used to cleave the vector into the 5 ′ and 3 ′ ends of the DNA of the present invention to form double strands. Cleave with restriction enzymes. Next, the mixture of the vector and the DNA fragment is ligated by allowing DNA ligase to act. The recombinant DNA thus obtained can be replicated indefinitely by appropriately introducing it into a host to form a transformant and culturing it.
[0031]
The recombinant DNA according to the present invention can be introduced into an appropriate host including E. coli, Bacillus subtilis, actinomycetes, yeast, plant cells, and animal cells. When the host is Escherichia coli, for example, the host may be cultured in the presence of recombinant DNA and calcium ions, while when the host is Bacillus subtilis, for example, the competent cell method or the protoplast method may be applied. Good. When the host is an animal cell, for example, the DEAE-dextran method or the electroporation method may be used. In order to clone the transformant, a hybridization method may be applied, or one that is cultured in a medium and produces L-asparaginase may be selected.
[0032]
When the transformant thus obtained is cultured in a medium, the polypeptide is produced outside or inside the cell or inside and outside the cell. The medium is usually a normal liquid medium supplemented with carbon sources, nitrogen sources, minerals, and micronutrients such as amino acids and vitamins as needed. As the carbon source, starch, starch hydrolysis can be used. Sugars such as food, glucose, fructose, sucrose, etc., and nitrogen sources include, for example, ammonia or ammonium salts, urea, nitrates, peptones, yeast extracts, defatted soybeans, corn steep liquor, meat extracts and other nitrogen-containing inorganic or organic substances Is mentioned. When the transformant is inoculated into such a medium and cultured for about 1 to 10 days under aerobic conditions such as aeration stirring while maintaining the nutrient medium at a temperature of 25 to 65 ° C. and pH 5 to 8, the polypeptide can be obtained. A culture containing is obtained. This culture may be used as a sensitive disease agent as it is. Usually, for example, prior to use, the cells or cells are disrupted by ultrasonic waves or cell wall lytic enzymes as necessary, and then filtered, centrifuged. The polypeptide is separated from the cell disruption or cell disruption by separation or the like and purified. In addition, for example, the culture supernatant obtained by removing cells or cells from the culture by filtration, centrifugation or the like is collected and purified. For purification, for example, salting out, dialysis, filtration, concentration, gel filtration chromatography, ion exchange chromatography, affinity to the supernatant or culture supernatant from which the insoluble components derived from bacterial cells or cell debris have been removed. Conventional methods commonly used in the art for purifying proteins such as chromatography, hydrophobic chromatography, isoelectric focusing and gel electrophoresis can be employed, and these methods may be combined as appropriate. Then, depending on the final use form, the purified polypeptide may be concentrated and lyophilized to make it liquid or solid.
[0033]
The method used here is described below based on experimental examples. For example, Jay Sambrook et al., “Molecular Cloning a Laboratory Manual” (1989), Cold. Also published in detail by Spring Harbor, Masami Matsumura, “Lab Manual Genetic Engineering” (1988), published by Maruzen.
[0034]
[Experiment 1]
<Expression of wild-type DNA derived from guinea pig and human>
[0035]
[Experimental Example 1-1]
<Expression of wild-type DNA derived from guinea pig>
[0036]
[Experimental Example 1-1 (a)]
<Preparation of guinea pig-derived wild-type DNA>
Wild-type DNA encoding L-asparaginase from guinea pig has been obtained by the same applicant. Special Kaihei 8-214885 public In the news Prepared according to the disclosed method. This DNA had the base sequence shown in SEQ ID NO: 15 in the sequence listing. Hereinafter, in the base sequence shown in SEQ ID NO: 15, a DNA encoding a polypeptide, that is, a DNA having a sequence consisting of the 20th to 1714th bases in the base sequence is referred to as “GPA / WT DNA”. A polypeptide having the amino acid sequence shown in SEQ ID NO: 15 in the sequence listing, which is an expression product of GPA / WT DNA, is hereinafter referred to as “guinea pig wild-type L-asparaginase”. In addition, in SEQ ID NO: 17 in the sequence listing, the amino acid sequence encoded by it is shown together with the base sequence of GPA / WT DNA.
[0037]
[Experimental Example 1-1 (b)]
<Preparation of recombinant DNA>
In a 0.5 ml reaction tube, 10 μl of 10 × PCR buffer, 1 μl of 25 mM dNTP mix, and 1 ng of wild-type DNA encoding L-asparaginase derived from guinea pig obtained in Experimental Example 1-1 (a) were used as a template. After adding appropriate amounts of oligonucleotides chemically synthesized based on sequences near the N-terminal and C-terminal in the amino acid sequence shown in SEQ ID NO: 15 in the Sequence Listing as sense primers and antisense primers to make 99.5 μl with sterile distilled water Then, 0.5 μl of 2.5 units / μl of Amplac DNA polymerase was added. The sense primer has a sequence of 5'-AATCTCGAGCCCACCCATGGCGCGCGCATCA-3 ', and M Kozak's "New Craik Acid Research" is located upstream of the portion encoding the N-terminus of the amino acid sequence shown in SEQ ID NO: 15 in the Sequence Listing. 15: 8125-8148 (1987), a common sequence in animal cells was added, and a restriction enzyme Xho I cleavage site was further added upstream. The antisense primer has a sequence of 5′-CTGCGGCCCCTTATCAGATGGCAGGCGGCAC-3 ′, and adds two start codons downstream of the portion encoding the C-terminal of the amino acid sequence shown in SEQ ID NO: 15 in the sequence listing, and further downstream And a sequence complementary to the sequence added with a restriction enzyme Not I cleavage site. In a conventional manner, PCR is performed by repeating 40 cycles of incubating the above mixture in the order of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes, and contains GPA / WT DNA. DNA was obtained. 25 ng of a DNA fragment of about 1.7 kbp obtained by cleaving this DNA with restriction enzymes Xho I and Not I was taken, and this was preliminarily cleaved with restriction enzymes XhoI and Not I. 10 ng, and then add the same volume of Takara Shuzo Ligation Kit Version 2 solution I to the previous DNA mixed solution, and then incubate at 16 ° C. for 2 hours to produce a replicable recombinant DNA “pCGPA / WT I got.
[0038]
Recombinant DNA pCGPA / WT was introduced into E. coli strain MC1061 / P3 manufactured by Invitrogen by the competent cell method, and the obtained transformant was L broth medium (pH 7.) containing 20 μg / ml ampicillin and 10 μg / ml tetracycline. 2) was inoculated and cultured with shaking at 37 ° C. for 18 hours. The transformant was collected by centrifuging the culture, and recombinant DNA pCGPA / WT was extracted by applying the usual alkali-SDS method. When analyzed by an automated sequencer using a fluorometer, pCGPA / WT contains GPA / WT DNA linked to a stop codon at its 3 'end, and GPA / WT DNA is 5' downstream of the CMV promoter. It was confirmed that they were linked from the end toward the 3 ′ end.
[0039]
For the expression of DNA in Experimental Example 1 and Experimental Example 2 described later, a system using COS-1 cells (ATCC CRL-1650), which is a monkey kidney-derived cell line, as hosts was used. Since this system is a transient expression system, the introduced DNA is stably maintained in the transformant, that is, not retained for several days, and the target peptide cannot be repeatedly produced using the transformant. There are drawbacks. However, when a vector having the SV40 viral origin of replication such as the aforementioned plasmid vector “pCDM8” is introduced into the cell, the number of copies per cell is temporarily 10 ^Five It is known that the level of the DNA increases, and this has the advantage that the analysis of the target DNA expression product is extremely easy.
[0040]
[Experimental Example 1-1 (c)]
<Introduction and expression of recombinant DNA into COS-1 cells>
The recombinant DNA pCGPA / WT prepared in Example 1-1 (b) was obtained from Frederick M. Osberg et al., “Current Protocol in Molecular Biology” (1987), John Wiley and Sons. -Introduced into COS-1 cells according to the DEAE-dextran method introduced in Ink Issuance, Chapters 9.2.1 to 9.2.3 and Chapters 9.2.5 to 9.2.6, Expressed. Specifically, first, put 2.5 ml of DME medium containing 10% (v / v) fetal calf serum into one hole of a 6-well multi-well plate “3046” made by Becton Dickinson Labware with a diameter of 3.5 cm, 1.8 × 10 ^Five Inoculated COS-1 cells and 5% (v / v) CO ₂ The cells were cultured at 37 ° C. in an incubator. On the next day, the culture supernatant was removed with an aspirator, and the cells were washed with DME medium containing 50 mM Tris-HCl (pH 7.4), and then 2.8 μg / ml pCGPA / WT, 50 mM Tris-HCl (pH 7.4). 2.5 ml of DME medium containing 0.4 mg / ml DEAE-dextran and 0.1 mM chloroquine was added to each well, and 5% (v / v) CO 2 was added. ₂ It left still at 37 degreeC in the incubator for 4 hours. Thereafter, the supernatant was removed, 2.5 ml of 10 mM phosphate buffer solution (hereinafter referred to as PBS) containing 10 (v / v)% DMSO was added per well and allowed to stand at room temperature for 2 minutes, and the supernatant was removed. The cells were washed with a DME medium containing 50 mM Tris-hydrochloric acid (pH 7.4), 2.5 ml of COS medium manufactured by Cosmo Bio was added, and 5% (v / v) CO 2 was added. ₂ The target DNA was expressed by culturing at 37 ° C. for 3 days in an incubator. In addition, the same experiment was also performed using plasmid vector pCDM8 as a control group.
[0041]
After culturing for 3 days, the above-mentioned incubator was allowed to stand at −80 ° C. and frozen, and then the operation of thawing at room temperature was repeated 3 times to disrupt the cells. Thereafter, the entire culture was transferred to a centrifuge tube, and insoluble components were removed as a precipitate by centrifugation to obtain a total soluble fraction. This was concentrated in a membrane, and the total soluble fraction derived from one well was adjusted to 0.5 ml and used for the subsequent analysis.
[0042]
[Experimental Example 1-1 (d)]
<Measurement of L-asparaginase activity>
L-asparaginase activity was expressed as an activity value (unit) measured as follows. Specifically, 50 μl of the test sample was dispensed into a 1.5 ml reaction tube, while L-asparagine was dissolved in 50 mM phosphate buffer (pH 7.0) so as to be 1.4 mg / ml. 200 μl was added per tube to make a reaction mixture. The reaction tube was allowed to stand at 37 ° C. for 0, 1, 2, 4, 6 and 16 hours, and then L-aspartic acid in the reaction mixture was measured with an amino acid analyzer. In parallel with this, a system using an E. coli-derived L-asparaginase preparation diluted to 1.0, 0.5, or 0.25 units / ml was provided and allowed to stand at 37 ° C. for 0 and 1 hour. -A calibration curve was prepared based on the increase amount of L-aspartic acid obtained from the aspartic acid measurement result. The increased amount of L-aspartic acid measured in the test sample system was interpolated into this calibration curve, and the activity value of the test sample was estimated. The activity value of the test sample with low activity was estimated from the measurement result in a system in which the reaction time was extended to 2 hours or more. One unit of L-asparaginase was defined as the amount that liberates 1 μmol of ammonia from L-asparagine per minute when reacted under the above conditions.
[0043]
This treatment was applied to each of all the soluble fractions obtained in Experimental Example 1-1 (c) to obtain 3.6 × 10 6. ^Five Expressed as the total amount of L-asparaginase activity detected in the total soluble fraction from COS-1 cells. As a result, the activity of guinea pig wild-type L-asparaginase was 0.083 units. It was not detected in the control group.
[0044]
[Experimental Example 1-1 (e)]
<Western blotting>
First, an anti-L-asparaginase antibody was prepared by the following method. That is, an oligopeptide having a sequence represented by Gly-Ser-Gly-Asn-Gly-Pro-Thr-Lys-Pro-Asp-Leu-Leu-Gln-Glu-Leu-Arg-Cys chemically synthesized according to a conventional method Keyhole, limpet, and hemocyanin were bound to the C-terminal of, and purified, and then rabbits were immunized according to a conventional method. After 6 immunizations at 2 week intervals, whole blood was collected and purified by 50% (w / v) ammonium sulfate salting out to obtain anti-L-asparaginase / rabbit antiserum. Next, the total soluble fraction obtained in Experimental Example 1-1 (c) according to the method reported by Yu K. Remli in “Nature”, Vol. 227, pages 680 to 685 (1970). 0.2 ml of the solution was subjected to 12.5% (w / v) SDS-polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) in the presence of a reducing agent, and all the migrated polypeptides were removed from the SDS-polyacrylamide gel. After being transferred to the nitrocellulose membrane, H Tobin uses the previous anti-L-asparaginase rabbit antiserum, "Proceeding of the National Academy of Sciences of the U.S. Western blotting was performed according to the method reported in A, Vol. 76, pages 4350-4354 (1979). Color development was based on an alkaline phosphatase color development system. Compared with the control, the band specifically stained with the sample was confirmed, and the stained band was compared with the molecular weight marker to determine the molecular weight per subunit of L-asparaginase. The molecular weight markers used were bovine serum albumin (67 kDa), ovalbumin (45 kDa), soybean trypsin inhibitor (20.1 kDa) and α-lactalbumin (14.4 kDa), which were stained with amide black. The total soluble fraction obtained in Experimental Example 1-1 (c) did not show a clear band.
[0045]
[Experimental Example 1-1 (f)]
<Measurement of molecular weight by gel filtration>
Next, 2 ml of the total soluble fraction derived from COS-1 cells obtained in Experimental Example 1-1 (c) was equilibrated with PBS, “High Road Superdex 200 Column” (inner diameter 16 mm). The native molecular weight of guinea pig wild-type L-asparaginase was examined by subjecting it to gel filtration column chromatography using × 60 cm) and examining the L-asparaginase activity of the eluted fraction. As molecular weight markers, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa) and ovalbumin (43 kDa) were used. As a result, the peak of L-asparaginase activity in the eluted fraction was observed at a position corresponding to a molecular weight of about 300 kDa.
[0046]
Since Western blotting did not show a clear band, the molecular weight of guinea pig wild-type L-asparaginase in a dissociated state could not be measured. The molecular weight in its native state was estimated to be about 300 kDa from the results of gel filtration. On the other hand, when guinea pig serum L-asparaginase is purified and the molecular weight in the native state is determined by gel filtration, it is estimated to be about 190 kDa. Incidentally, the molecular weight in the dissociated state by SDS-PAGE is estimated to be about 43 kDa. Meanwhile, by the same applicant Special Kaihei 8-214885 public In the news The three partial amino acid sequences of the disclosed guinea pig serum L-asparaginase are found in a region consisting of amino acid residues 10 to 236 in the amino acid sequence of guinea pig wild-type L-asparaginase. In addition, E-Harms et al., “Febs Letter”, Vol. 285, pp. 55 to 58 (1991), proposed from the experimental results using L-asparaginase derived from Escherichia coli, etc., are essential for L-asparaginase activity. The amino acid sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing are the 16th to 19th amino acid residues and the 114th to 118th amino acid residues on the amino acid sequence of guinea pig wild-type L-asparaginase, respectively. Matches the base sequence. From these results and the results shown in Experimental Example 1-1, the present inventor, for guinea pig wild-type L-asparaginase, comprises the first to 400th amino acid residues in the amino acid sequence or the amino acid residues before and after that. However, it was speculated that it would be essential for its activity expression. Therefore, in Experimental Example 2-1, in order to examine the activity of the C-terminal deletion mutant which is a guinea pig-derived L-asparaginase homologue, the properties and properties of the expression product of the guinea pig-derived DNA homologue are tested.
[0047]
[Experimental Example 1-2]
<Expression of human-derived wild-type DNA>
Wild-type DNA encoding human L-asparaginase has been obtained by the same applicant. Special Kaihei 8-214885 public In the news Prepared according to the disclosed method. This DNA had the base sequence shown in SEQ ID NO: 16 in the sequence listing. Hereinafter, a region encoding a polypeptide in the base sequence shown in SEQ ID NO: 16, that is, a DNA having a sequence consisting of the 93rd to 1811th bases in the base sequence is referred to as “HA / WT DNA”. In addition, a polypeptide having the amino acid sequence listed in SEQ ID NO: 16, which is an expression product of HA / WT DNA, may hereinafter be referred to as “human wild type L-asparaginase”. In addition, in SEQ ID NO: 18 in the sequence listing, the amino acid sequence encoded by the base sequence of HA / WT DNA is also shown.
[0048]
PCR was performed under the same conditions as in Experimental Example 1-1 (b) except for the template, sense primer, and antisense primer. The template is a wild-type DNA encoding human-derived L-asparaginase obtained in Experimental Example 1-2, the sense primer is an oligonucleotide having a sequence of 5'-AATCTCGAGCCCCATGGCGCGCGCGTG-3 ', and the antisense primer is a sequence of 5' -An oligonucleotide that is CTGCGGCCCCTTATCAGACACCAGGCAGCAC-3 '. The resulting amplified DNA was subsequently treated in the same manner as in Experimental Example 1-1 (b) to prepare a recombinant DNA “pCHA / WT”. After confirming the sequence in the same manner, it was introduced into COS-1 cells, expressed, and analyzed in the same manner as in Experimental Example 1-1.
[0049]
In contrast to guinea pig wild-type L-asparaginase, human wild-type L-asparaginase could not detect activity in this experimental system. As one of the causes, for example, human wild-type L-asparaginase was considered to have a lower specific activity than guinea pig wild-type L-asparaginase. Therefore, in the following Experimental Example 2-2, the properties and properties of the expression products of human-derived DNA homologues are tested.
[0050]
[Experimental example 2]
<Expression of DNA homologues derived from guinea pig and human>
[0051]
[Experimental Example 2-1]
<Expression of DNA homologue from guinea pig>
A DNA homologue of a guinea pig-derived wild type DNA in which the base sequence at a specific position was replaced with a stop codon was prepared as follows. That is, in the base sequence shown in SEQ ID NO: 17 in the sequence listing, a DNA in which the sequence consisting of the 1090th to 1092th bases is replaced with a stop codon, and a DNA in which the sequence consisting of the 1012th to 1014th bases is replaced with a stop codon Was prepared by applying the PCR method. Except for the sequence of the antisense primer, PCR was carried out under the same conditions as in Experimental Example 1-1 (b). The sequences of the antisense primers used for each DNA preparation were 5′-CTGCGGCCCCTTATCATGCCGGTGGGCAGGTGT-3 ′ and 5′-CTGCGGCCGCTTATCAGCCCAACACGGTAGGA-3 ′. The resulting amplified DNA was subsequently treated in the same manner as in Experimental Example 1-1 (b) to prepare recombinant DNAs “pCGPA / D364stp” and “pCGPA / L338stp”. As a result of confirming the sequence in the same manner, pCGPA / D364stp and pCGPA / L338stp each encode a sequence comprising the first to 363th amino acid residues and the first to 337th amino acid residues in guinea pig wild-type L-asparaginase, respectively. And a stop codon present without intervening sequence at each 3 ′ end. Hereinafter, the DNAs encoding these polypeptides are referred to as “GPA / D364stp DNA” and “GPA / L338stp DNA”, respectively. GPA / D364stp DNA and GPA / L338stp DNA were ligated downstream from the CMV promoter in the direction from the 5 'end to the 3' end. Hereinafter, the expression products of these DNAs are sometimes referred to as “guinea pig L-asparaginase homologues”.
[0052]
These recombinant DNAs were similarly tested after being introduced into COS-1 cells according to Experimental Example 1-1. As a control, the recombinant DNAs pCGPA / WT and pCDM8 prepared in Experimental Example 1-1 (b) were similarly treated and tested. The results are shown in Table 1.
[0053]
[Table 1]

[0054]
As shown in Table 1, among the wild-type DNA derived from the above guinea pigs and homologues thereof, GPA / WT DNA and GPA / D364stp DNA expression products showed activity, but GPA / L338stp DNA expression product No activity was detected. This means that it is sufficient for the guinea pig-derived L-asparaginase to have sufficient activity in the amino acid sequence of the wild-type L-asparaginase. Suggests. The sequence consisting of the first to 363th amino acid residues is shown in SEQ ID NO: 4 in the sequence listing. The base sequence of the DNA encoding this is shown in SEQ ID NO: 10 in the sequence listing. The amino acid sequence of guinea pig wild-type L-asparaginase is shown in SEQ ID NO: 5 in the sequence listing.
[0055]
[Experimental example 2-2]
<Expression of DNA homologues derived from human>
A DNA homologue was prepared by substituting the base sequence at a specific position of human-derived wild-type DNA with a stop codon or a codon for another amino acid. First, a DNA homologue in which the sequence consisting of the 1096th to 1098th bases in the base sequence shown in SEQ ID NO: 18 in the sequence listing was replaced with a stop codon was prepared by applying the PCR method. That is, except for the template, sense primer, and antisense primer, PCR was performed under the same conditions as in Experimental Example 1-1 (b). The template is a wild-type DNA encoding human-derived L-asparaginase obtained in Experimental Example 1-2, the sense primer is an oligonucleotide whose sequence is 5'-AATCTCGAGCCCCATGGCGCGCGCGTG-3 ', and the antisense primer is 5' -An oligonucleotide which is CTGCGGCCGCTCATTACACCGAGGGTGGCGT-3 '. As a result, the amplified DNA was treated according to Experimental Example 1-1 to prepare a recombinant DNA “pCHA / E366stp”, and the sequence was confirmed. pCHA / E366stp includes DNA encoding a sequence consisting of amino acid residues 1 to 365 in the amino acid sequence shown in SEQ ID NO: 16 in the sequence listing, and a stop codon present at its 3 'end without an intervening sequence Met. Hereinafter, the DNA of this coding part is referred to as “HA / E366stp DNA”. HA / E366 stpDNA was ligated downstream from the CMV promoter in the 5 'to 3' end direction.
[0056]
To replace a codon at a specific position of DNA with a codon for another amino acid, Robert M. Horton et al., “Methods in Enzymology”, Academic Press, Vol. 217, pp. 270-279 ( (1993) and the overlap extension method introduced. The outline is shown in FIG. 1 and described below. First, a mutation primer A and a mutation primer S that are complementary to each other are prepared by replacing the base at the position where the mutation is to be introduced with another target base. Here, the mutation primer A is an antisense strand, and the mutation primer S is a sense strand. On the other hand, a primer set capable of amplifying the entire region of the target DNA, that is, a 5 ′ end primer and a 3 ′ end primer is prepared. Here, the 5 ′ end primer is a sense strand, and the 3 ′ end primer is an antisense strand. Second, normal PCR is performed using the DNA of the base sequence of the group as a template and the 5 ′ end primer and the mutation primer A. In parallel with this, normal PCR is performed using the same DNA as a template and the previous 3'-end primer and mutation primer S (first-stage PCR). The two DNAs amplified by the third and first stage PCR, the 5 ′ end primer and the 3 ′ end primer used in the first stage PCR are mixed in the same reaction tube, and PCR is performed (second stage PCR). The two DNA fragments amplified in the first stage PCR are used to generate DNA with mutations introduced as templates and primers, and the 5 ′ end primer and the 3 ′ end primer are used to amplify the DNA with mutations introduced. Used as a primer. By this method, DNA into which seven kinds of base substitutions were introduced, that is, seven kinds of DNA homologues were prepared. Table 2 summarizes the contents of the seven types of base substitutions and the changes in the amino acid sequence that accompanies them. Table 3 summarizes the sequences of template DNA, mutation primer A, and mutation primer S used for the preparation of these seven DNA homologues. On the other hand, the 5′-end primer and the 3′-end primer used for the preparation of the seven DNA homologues were respectively the sense primer and the antisense used in the preparation of pCHA / E366stp previously shown in this Experimental Example 2-2. Identical to the primer.
[0057]
[Table 2]

[0058]
[Table 3]

[0059]
The human-derived DNA homologue obtained here was treated according to Experimental Example 1-1, and recombinant DNAs “pCHA / MUT1”, “pCHA / MUT2”, “pCHA / MUT3”, “pCHA / MUT4”, “pCHA / “MUT5”, “pCHA / MUT6” and “pCHA / MUT7” were obtained. Hereinafter, the expression product of the above DNA homolog obtained in Experimental Example 2-2 may be referred to as “human L-asparaginase homolog”. Similarly, after confirming the sequence, it was introduced into COS-1 cells for expression and tested. As a control group, pCHA / WT and pCDM8 obtained in Experimental Example 1-2 were similarly treated and tested. In Experimental Example 2-2, the signal intensity of the band detected by Western blotting was quantified by densitometry as a reference for quantitative comparison of each expression product. The above results are shown in Table 4.
[0060]
[Table 4]

[0061]
The results in Table 4 show that the specific activity of human-derived L-asparaginase is higher than that of guinea pig-derived L-asparaginase, even if it is wild-type or C-terminal deletion mutant (HA / E366stp DNA expression product) which is one of its homologues. Suggests low. On the other hand, among the point mutants in which some of the original amino acid sequence of wild-type L-asparaginase derived from human is substituted with other amino acids, the specific activity increases to such a degree that it can be detected. It also shows that there is. The human-derived DNA homologs HA / MUT1 DNA, HA / MUT2 DNA, HA / MUT3 DNA, and HA / MUT5 DNA, in which the expression product has been confirmed to exhibit at least detectable activity, are shown in SEQ ID NOs: The polypeptides having the nucleotide sequences shown in 11, 12, 13, and 14 have the amino acid sequences shown in SEQ ID NOs: 6, 7, 8, and 9 in the sequence listing, respectively.
[0062]
From the above experimental results, in order to exhibit a L-asparaginase activity at a level at which a polypeptide derived from a mammal can be detected at least in the expression system / activity measurement system used in Experimental Examples 1 and 2, a conventionally known arrangement may be used. In addition to the amino acid sequences shown in SEQ ID NOS: 1 and 2 in the table, for example, it was found that it is necessary to have the amino acid sequence shown in SEQ ID NO: 3 (however, the amino acid shown with the symbol “Xaa” is glutamine Or arginine). Incidentally, guinea pig wild-type L-asparaginase is a portion consisting of residues 298 to 302 in the amino acid sequence. In It has this arrangement. Examples of the polypeptide having all the amino acid sequences shown in SEQ ID NOS: 1 to 3 in the sequence listing are derived from guinea pigs, polypeptides having the amino acid sequences shown in SEQ ID NO: 4 to 5 in the sequence listing, and humans. Examples include polypeptides having the amino acid sequences shown in SEQ ID NOs: 6 to 9 in the sequence listing.
[0063]
Based on the above findings, the present inventors have invented a mammal-derived polypeptide having L-asparaginase activity. The invention will now be described on the basis of examples which are all conventional in the field. Of course, the method for carrying out the invention is not limited to these.
[0064]
Example A-1
<Wild-type polypeptide derived from guinea pig having L-asparaginase activity>
[0065]
Example A-1 (a)
<Preparation of transformant>
In a 0.5 ml reaction tube, 10 μl of 10 × PCR buffer, 1 μl of 25 mM dNTP mix, 1 ng of the recombinant DNA pCGPA / WT obtained in Experimental Example 1-1 as a template, and 5 ′ end of GPA / WT DNA Add an appropriate amount of the oligonucleotide chemically synthesized based on the sequence of the 3 ′ end as a sense primer or an antisense primer, make 99.5 μl with sterilized distilled water, and then add 0.5 μl of 2.5 units / μl ampli-tack DNA polymerase. It was. The sense primer has a sequence of 5'-GCGAATTCATGGCGCGCGCATCA-3 'and has a restriction enzyme Eco RI cleavage site added upstream of the 5' end of GPA / WT DNA. The antisense primer has a sequence of 5'-GCAAGCTTTTGAGATGGCAGGCGGCAC-3 'and is complementary to a sequence in which a stop codon is added downstream of the 3' end of GPA / WT DNA and a restriction enzyme HindIII cleavage site is further added downstream thereof. Of a typical array. In a conventional manner, PCR was performed by amplifying DNA by repeating 40 cycles of incubating the mixture in the order of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes. This DNA was cleaved with restriction enzymes Eco RI and Hin d III to obtain an Eco RI-Hind dIII fragment of about 1.7 kbp. Take 25 ng of this DNA fragment, add 10 ng of Pharmacia plasmid vector “pKK223-3” previously cut with restriction enzymes Eco RI and HindIII, and then add Solution I of Takara Shuzo Ligation Kit Version 2 first. After adding the same volume as that of the DNA mixture solution, the recombinant DNA “pKGPA / WT” capable of replication was obtained by incubating at 16 ° C. for 2 hours.
[0066]
Recombinant DNA pKGPA / WT was introduced into E. coli strain JM105 produced by Pharmacia by the competent cell method, and the transformant “J-GPA / WT” obtained here was added to L broth medium (pH 7.2) containing 50 μg / ml ampicillin. ) And cultured with shaking at 37 ° C. for 18 hours. The transformant was collected by centrifuging the culture, and the recombinant DNA pKGPA / WT was extracted by applying the usual alkali-SDS method. By analyzing with an automatic sequencer using a fluorometer, in this pKGPA / WT, as shown in FIG. 2, the GPA / WT DNA having the base sequence shown in SEQ ID NO: 17 in the sequence listing is located at the 5 ′ end downstream of the tack promoter. It was confirmed that they were linked in the 3 ′ end direction. It was also confirmed that there was a stop codon without an intervening sequence on the 3 ′ end side of GPA / WT DNA.
[0067]
Example A-1 (b)
<Manufacture of polypeptides>
The transformant J-GPA / WT was inoculated into L broth medium (pH 7.2) containing 50 μg / ml ampicillin and cultured with shaking at 37 ° C. for 18 hours. Next, 18 l of fresh L broth medium was placed in a 30 l jar fermenter, the seed culture previously cultured was inoculated at a rate of 1% (v / v), and cultured at 37 ° C. with aeration and stirring. A portion of the culture is placed in a 1 cm thick cuvette, cultured while measuring the absorbance at a wavelength of 650 nm, and when the absorbance reaches about 1.5, IPTG is added to a final concentration of 0.1 mM. Cultured for 5 hours. Thereafter, the cells recovered from the culture by centrifugation are suspended in a mixed solution (pH 7.2) containing 139 mM sodium chloride, 7 mM disodium hydrogen phosphate and 3 mM sodium dihydrogen phosphate, and sonicated by a conventional method. The cells were crushed, and the crushed cells were centrifuged to collect the supernatant.
[0068]
To this supernatant was added ammonium sulfate to 50% (w / v) under ice-cooling, and the mixture was uniformly dissolved, allowed to stand for a while and centrifuged, and the precipitate was collected. This precipitate is dissolved in 20 mM Tris-HCl buffer (pH 8.0), dialyzed into the same buffer, loaded onto a Pharmacia “Q Sepharose F. F. column” equilibrated with the same buffer, and the same buffer. After thoroughly washing with the solution, a 20 mM Tris-HCl buffer (pH 8.0) was passed through a concentration gradient of 0 to 0.5 M sodium chloride. The fraction eluted at a sodium chloride concentration of about 0.1 to 0.3 M was collected, and the solvent was exchanged into 10 mM sodium phosphate buffer (pH 7.5) while concentrating the membrane. Sigma “L-asparagine agarose” equilibrated with the same buffer was loaded, washed with the same buffer, and eluted with 10 mM sodium phosphate buffer (pH 7.5) containing 0.5 M sodium chloride. The elution fraction was concentrated in a membrane and loaded onto a Pharmacia “Hiload Superdex 200 column” equilibrated with Tris-HCl buffer solution (pH 8.0) containing 10% (v / v) glycerol. When an elution fraction of about 300 kDa was collected, a purified polypeptide having a purity of 90% or more was obtained at a yield of about 0.1 mg / ml per culture.
[0069]
Example A-1 (c)
<Physical and chemical properties>
The purified polypeptide was analyzed as follows to clarify its physicochemical properties. The native molecular weight of the purified polypeptide was determined by gel filtration according to Experimental Example 1-1 (e). As a result, the peak of L-asparaginase activity in the eluted fraction was observed at a position corresponding to a molecular weight of about 300 kDa. The molecular weight of the purified polypeptide in the dissociated state was determined by SDS-PAGE shown in Experimental Example 1-1 (e). As a result, a main band was observed at a molecular weight of 50 ± 10 kDa. This result indicates that the purified polypeptide forms a multimer in its native state. Taking into account the measurement errors of the two methods and the native form of the known L-asparaginase other than mammals including E. coli, all in the native state, this result is a purified polypeptide. Is considered to indicate that a tetramer is formed. In addition, as a result of subjecting this purified polypeptide to the method shown in Experimental Example 1-1 (d), it was confirmed that it had L-asparaginase activity.
[0070]
Example A-2
<Wild-type polypeptide derived from guinea pig having L-asparaginase activity>
[0071]
Example A-2 (a)
<Preparation of transformant>
An outline is shown in FIG. First, PCR was performed under the same conditions as in Example A-1 (a) except for the sequences of the sense primer and the antisense primer. The sequence of the sense primer was 5'-GTGAATTCGGAGGTTCAGATGCGCGCGCATCA-3 ', and the sequence of the antisense primer was 5'-CTGCGGCCGCTCAGATGGCAGGCGGCAC-3'. The amplified DNA was cleaved with restriction enzymes Eco RI and Not I to obtain an Eco RI-Not I fragment of about 1.7 kbp. 70 ng of this DNA fragment, 50 ng of Pharmacia plasmid vector “pBPV” previously cut with restriction enzymes Xho I and Not I, and 4 ng of the following 4 nucleotide oligonucleotides were mixed as linkers. . The sequence of the first oligonucleotide is 5'-TCGAGCCCACCCATGAAGTGTTCGGTGGGTTTATT-3 ', the second is 5'-TTCTTCCTGATGGCCGTAGTGACAGGAGGTG-3', the third is 5'-AATTCACTCCTGTCACTAG-3 '5'-AGAAATAACCCACGAACACTTTCATGGTGGC-3'. The oligonucleotides used as linkers were all synthesized according to a conventional method and then purified by ethanol precipitation after acting on Pharmacia T4 polynucleotide kinase. To this DNA mixed solution, an equal volume of Takara Shuzo Ligation Kit Version 2 Solution I was added and kept at 16 ° C. for 2 hours to obtain a replicable recombinant DNA “pBIgGPA / WT”.
[0072]
Recombinant DNA pBIgGPA / WT was introduced into E. coli strain HB101 by the competent cell method, and the resulting transformant was inoculated into L broth medium (pH 7.2) containing 50 μg / ml ampicillin and shaken at 37 ° C. for 18 hours. Cultured at last. The culture was centrifuged to collect the cells, and the recombinant DNA pBIgGPA / WT was extracted by applying a normal alkali-SDS method. When the sequence was analyzed by an automatic sequencer, it was confirmed that this pBIgGPA / WT had a structure as shown in FIG. That is, D.F. Stern is “Science” downstream of the transcriptional regulatory region consisting of the Moloney murine sarcoma virus long terminal repeat-derived enhancer (Emsv) and the mouse metallothionein I gene-derived promoter (Pmti). Volume 235, pp. 321 to 324 (1987). DNA encoding the peptide part containing the immunoglobulin secretion signal sequence, Ig sec DNA, is linked, and further downstream in the same reading frame, GPA / WT DNA was ligated from the 5 'end to the 3' end. It was also confirmed that there was a stop codon at the 3 'end of GPA / WT DNA without an intervening sequence.
[0073]
This recombinant DNA pBIgGPA / WT was introduced into a mouse-derived cell line C127 (ATCC CRL-1616) using Lipofectin reagent manufactured by Life Technologies according to the attached protocol. The transformant introduced with the recombinant DNA was selected as a first step based on loss of growth control ability, that is, focus-forming ability. As a second step, cells in the vicinity including the focus were collected with sterile filter paper, and the target transformant was made into a single cell based on the ability to produce L-asparaginase activity by an ordinary limiting dilution method. Thus, a transformant “C-GPA / WT” was obtained.
[0074]
Example A-2 (b)
<Manufacture of polypeptides>
Transformant C-GPA / WT was first seeded with a 6-well multi-centrifuge manufactured by Becton Dickinson Labware with a diameter of 3.5 cm to which 2.5 ml of DME medium containing 10% (v / v) fetal calf serum was added. One well of the well plate “3046” was inoculated and cultured to confluence. Cells were detached from the cells by trypsin treatment, and a portion thereof was used as seed cells to inoculate into 6 holes of another new 6-hole plastic plate to which the same medium had been added. Repeat the same procedure while expanding the incubator in order to increase the number of cells to 150 cm. ² The transformant was subjected to normal continuous culture using 50 culture flasks. Finally, 100 l of the culture supernatant was collected, and the ammonium sulfate precipitation and precipitation solution Kew Sepharose FF column was used in the same manner as in the method for treating the supernatant of the disrupted bacterial cells of Example A-1 (b). Chromatography, chromatography using L-asparagine agarose of the eluted fraction, and chromatography using Hiload Superdex 200 column of the eluted fraction. As a result, a purified polypeptide having a purity of 90% or more was obtained at a yield of about 1 μg / ml per culture.
[0075]
Example A-2 (c)
<Physical and chemical properties>
When the physicochemical properties of the obtained purified polypeptide are examined in the same manner as in Example A-1 (c), it should exhibit the same properties as the purified polypeptide obtained in Example A-1 (b). Was confirmed.
[0076]
Example A-3
<Polypeptide homologue derived from human and guinea pig having L-asparaginase activity>
[0077]
Example A-3 (a)
<Preparation of transformant>
By performing PCR under the same conditions as in Example A-1 (a) except for the template, sense primer and antisense primer, and treating the resulting DNA in the same manner as in Example A-1 (a) Recombinant DNAs “pKGPA / D364stp”, “pKHA / MUT1”, “pKHA / MUT2”, “pKHA / MUT3” and “pKHA / MUT5” were obtained. Table 5 summarizes the name of the template DNA used for the preparation of each recombinant DNA, and the base sequences of the sense primer and the antisense primer. The base sequences were analyzed in the same manner as in Example A-1 (a), and the structures of these recombinant DNAs The confirmed. The structures of these recombinant DNAs are shown in FIGS.
[0078]
[Table 5]

[0079]
The recombinant DNA obtained here was similarly treated according to Example A-1 (a), and transformants “J-GPA / D364stp”, “J-HA / MUT1”, “J-HA / MUT2”, “J-HA / MUT3” and “J-HA / MUT5” were obtained.
[0080]
Example A-3 (b)
<Manufacture of polypeptides>
These five types of transformants were cultured, as in Example A-1 (b), microbial disruption, ammonium sulfate precipitation of the microbial disruption, and chromatography using a Kew Sepharose FF column of the precipitate solution. And chromatography of the eluted fractions using L-asparagine agarose. The resulting elution fraction was subjected to membrane concentration in the same manner as in Example A-1 (b), and then subjected to chromatography using a high-load superdex 200 column, and an elution fraction of about 140 kDa was obtained. When collected, a purified polypeptide with a purity of 90% or more was obtained at a yield of about 0.1 mg / ml per culture. These purified polypeptides were analyzed by the method of Example A-1 (c) to clarify physicochemical properties. The results are shown in Table 6 together with the results obtained in Example A-1.
[0081]
[Table 6]

[0082]
The results shown in Table 6 indicate that each wild-type polypeptide or polypeptide homologue purified by expressing Escherichia coli as a host exhibits L-asparaginase activity. These polypeptides are also shown to form a tetramer.
[0083]
Example A-4
<Polypeptide homologue derived from human and guinea pig having L-asparaginase activity>
[0084]
Example A-4 (a)
<Preparation of transformant>
PCR was performed under the same conditions as in Example A-1 (a) except for the template, sense primer or antisense primer. The obtained DNA was ligated under the same conditions using the same linker as used in Example A-2 (a), so that recombinant DNA “pBIgGPA / D364stp”, “pBIgHA / MUT1”, “pBIgHA” were used. / MUT2 ”,“ pBIgHA / MUT3 ”and“ pBIgHA / MUT5 ”. Table 7 summarizes the names of template DNAs used in PCR for the preparation of each recombinant DNA and the base sequences of the sense primer and antisense primer. Base sequences were analyzed in the same manner as in Example A-1 (a), and the structures of these recombinant DNAs were confirmed. The structures of these recombinant DNAs are shown in FIGS.
[0085]
[Table 7]

[0086]
The recombinant DNA obtained here was similarly treated in the same manner as in Example A-2 (a), whereby transformants “C-GPA / D364stp”, “C-HA / MUT1”, “C- “HA / MUT2”, “C-HA / MUT3” and “C-HA / MUT5” were obtained.
[0087]
Example A-4 (b)
<Manufacture of polypeptides>
[0088]
These five types of transformants were cultured in the same manner as in Example A-2 (b), and the culture supernatant was precipitated with ammonium sulfate and precipitated solution in the same manner as in the cell disruption method in Example A-1 (b). The chromatography was performed in the order of chromatography using a Kew Sepharose F. F column and chromatography using L-asparagine agarose gel of the eluted fraction. The resulting elution fraction was subjected to membrane concentration in the same manner as in Example A-1 (b), and then subjected to chromatography using a high-load superdex 200 column, and an elution fraction of about 140 kDa was obtained. When collected, a purified polypeptide having a purity of 90% or more was obtained at a yield of about 1 μg / ml per culture. These purified polypeptides were analyzed by the method of Example A-1 (c) to clarify physicochemical properties. Table 8 shows the results together with the results obtained in Example A-3.
[0089]
[Table 8]

[0090]
The results shown in Table 8 indicate that each wild-type polypeptide or polypeptide homologue purified by expressing animal cells as a host exhibits L-asparaginase activity. It is also shown that these polypeptides form a tetramer.
[0091]
As described above in Example A, all of the polypeptides of the present invention exhibit L-asparaginase activity. Therefore, when administered to humans, the sensitive disease agent of the present invention hydrolyzes L-asparagine in the body and exerts an effect on the treatment and prevention of L-asparaginase sensitive diseases. Sensitive diseases as used in the present invention mean all diseases caused by the presence of L-asparagine-dependent tumor cells. Specifically, for example, acute leukemia, acutely transformed chronic leukemia, T cell leukemia, etc. And malignant lymphomas such as leukemia, non-Hodgkin's disease and Hodgkin's disease. Thus, the sensitive disease agent of the present invention has a use as an antitumor agent for treating / preventing a sensitive disease as described above. Depending on the dosage form and the type and nature of the sensitive disease, the sensitive disease agent of the present invention is usually prepared in a liquid, paste or solid form, and the polypeptide is 0.000001 to 100% (w / w). Desirably, it contains 0.0001 to 100% (w / w).
[0092]
The sensitive disease agent of the present invention is not only in the form of the polypeptide alone, but also in the polypeptide and other physiologically acceptable, for example, base, excipient, solubilizer, buffer, stabilizer, As needed, the form as a composition with 1 type, or 2 or more types chosen from other physiologically active substance thru | or another chemical | medical agent is also included. Specific examples of bases, excipients, solubilizers, buffers, and stabilizers include, for example, edited by Japan Pharmaceutical Additives Association, “Pharmaceutical Additives Dictionary” (1994), published by Yakuji Nippo. Examples include those described in the “Additives Supplement Supplement 1995” (1995), published by Yakuji Nippo. Specific examples of other physiologically active substances or other drugs include, for example, interferon-α, interferon-β, interferon-γ, interleukin 1, interleukin 2, interleukin 3, TNF-α, TNF-β, GM-CSF, carbocon, cyclophosphamide, aclarubicin, thiotepa, busulfan, ancitabine, cytarabine, fluorouracil, tetrahydrofurylfluorouracil, methotrexate, actinomycin D, chromomycin A3, daunorubicin, doxorubicin, bleomycin, mercaptopurine C, predomycin C , Vincristine, vinblastine, colloidal gold, krestin, picibanil, lentinan, and Maruyama vaccine.
[0093]
Furthermore, the susceptible disease agent of the present invention also includes a dosage unit form of the drug, which means that the polypeptide is administered at a dose per dose or a positive multiple thereof (up to 4 times). ) Or a fraction thereof (up to 1/40) means a drug in a physically separate unitary dosage form suitable for dosing. Examples of the drug in such a dosage form include injections, liquids, powders, granules, tablets, capsules, sublinguals, eye drops, nasal drops, suppositories and the like.
[0094]
The sensitive disease agent of this invention is effective for the treatment and prevention of sensitive diseases in both cases, whether orally or parenterally. Although it depends on the type and symptoms of the sensitive disease, specifically, about 0.1 μg to 500 mg / dose, preferably about 0.1 to 100 mg / dose per adult, while observing the patient's symptoms and the course after administration. The polypeptide may be orally administered at a dose of 1 to 4 times / day or 1 to 7 times / week for 1 day to 1 year, or parenterally intradermally, subcutaneously, intramuscularly or intravenously. Moreover, as another form of the sensitive disease agent of this invention, the form which applied gene therapy is mentioned, for example. That is, by administering a transformant in which a DNA encoding the polypeptide of the present invention is appropriately introduced into a host and producing the polypeptide of the present invention in a living body of a patient with a sensitive disease, it is equivalent to the above dosage form. Demonstrate the effect. General procedures for carrying out gene therapy are, for example, edited by Takashi Shimada, Izumi Saito, Takaya Ozawa, "Experimental Medicine Separate Bio Manual UP Series Basic Technology of Gene Therapy" (1996), published by Yodosha. Is described in detail.
[0095]
Next, the biological activity of the polypeptide of the present invention will be described based on Experimental Example 3, and the safety of the polypeptide of the present invention will be described based on Experimental Example 4.
[0096]
[Experiment 3]
<Bioactivity>
[0097]
[Experimental Example 3-1]
<Anti-tumor cell effect in vitro>
[0098]
Human histiocytic lymphoma cell line U937 (ATCC No. CRL-1593) and human lymphoblast-derived cell line Molt4 (ATCC No. CRL-1582) were previously prepared in RPMI 1640 medium containing 10% (v / v) fetal bovine serum. Successor Subcultured. Cells are collected from each cell culture system in the logarithmic growth phase by centrifugation and 2 × 10 2 in the same medium. ⁵ The cell concentration was adjusted to cell number / ml, and the cell suspension was inoculated into a 24-well multi-well plate “3047” plate manufactured by Becton Dickinson Labware in a total of 13 wells at 1 ml per well. To this, dilutions of 12 kinds of purified polypeptides prepared in Examples A-1 to A-4 with PBS were respectively added, and 5% (v / v) CO 2 was added. ₂ The cells were cultured at 37 ° C. for 72 hours in an incubator. The final concentration of each purified polypeptide was 1 unit / ml as L-asparaginase activity. As a control group, a system in which an equal volume of PBS was added and cultured in the same manner was provided. After the culture, the cells were collected, dead cells were stained with trypan blue, and the cell viability of the purified polypeptide addition system was compared with the control group. As a result, the system to which any of the purified polypeptides was added showed a significantly lower cell viability than the control group. This indicates that the purified polypeptides obtained in Examples A-1 to A-4 all have cytotoxicity against U937 and Molt4.
[0099]
[Experimental Example 3-2]
<Anti-tumor cell effect in vivo>
[0100]
A mouse lymphoma cell line 6C3HED registered at the Center for Medical Cell Resources, Institute of Aging Medicine, Tohoku University is used for 1 × 10 ⁷ By subcutaneous injection in the flank every 8 days per animal / animal Successor C3H mice that had been transplanted were used as model mice. The purified polypeptide obtained in Examples A-1 to A-4 was intravenously administered at 400 units / animal to the model mice every day from day 4 to day 7 after transplantation of the cells. . The size of the tumor on the 4th and 8th day after transplantation was observed with the naked eye. The purified polypeptide was administered after being diluted with a 0.15 M sodium chloride solution and then filtered through a membrane filter manufactured by Millipore having a pore diameter of 0.45 μm. As a control group, a system similarly treated with a 0.15 M sodium chloride solution was provided. As a result, clear enlargement of the tumor was observed in the control group, whereas clear regression or disappearance of the tumor was observed in the system administered with the purified polypeptide. This indicates that all of the purified polypeptides prepared in Examples A-1 to A-4 are capable of healing tumors in model mice.
[0101]
[Experimental Example 4]
<Acute toxicity test>
The purified polypeptides prepared in Examples A-1 to A-4 were separately administered to 8-week-old mice by transdermal, oral or intraperitoneal injection according to a conventional method. As a result, LD50 of these purified polypeptides was about 100 mg / kg or more by any route of administration. This confirms that the polypeptide of the present invention is safe as a pharmaceutical premised on administration to humans.
[0102]
Hereinafter, the sensitive disease agent of the present invention will be described with reference to examples.
[0103]
Example B
<Sensitive disease agent>
[0104]
Example B-1
<Liquid>
The purified polypeptides obtained in Examples A-1 to A-4 were dissolved separately in physiological saline containing 1% (w / v) human serum albumin as a stabilizer so as to be 0.1 mg / ml. The solution was sterilized by microfiltration according to a conventional method.
[0105]
All products have excellent stability and are useful as injections, eye drops, and nasal drops for the treatment and prevention of malignant tumors, acute leukemias, malignant lymphomas, chronic leukemia transformation, and sensitive diseases including T-cell leukemia It is.
[0106]
Example B-2
<Liquid>
Each of the purified polypeptides obtained in Examples A-1 to A-4 was dissolved separately in physiological saline containing 1% (w / v) glycerin as a stabilizer so as to be 0.1 mg / ml. Sterilized by microfiltration according to the method to obtain a solution.
[0107]
All products are excellent in stability as malignant tumors, acute leukemias, malignant lymphomas, chronic leukemia blast crisis, injections, eye drops, and nasal drops for treating and preventing sensitive diseases including T cell leukemia Useful.
[0108]
Example B-3
<Dry injection>
As a stabilizer, the purified polypeptides obtained in Examples A-1 to A-4 were separately dissolved in physiological saline containing 1% (w / v) of purified gelatin so as to be 50 mg / ml. The mixture was sterilized by microfiltration according to the method, dispensed in 1 ml portions into vials, freeze-dried and sealed.
[0109]
Each product has excellent stability and is useful as a dry injection for treating and preventing malignant tumors, acute leukemias, malignant lymphomas, chronic leukemias, and sensitive diseases including T cell leukemia.
[0110]
Example B-4
<Ointment>
In sterilized distilled water, carboxyvinyl polymer “Hibiswako 104” and high-purity trehalose manufactured by Wako Pure Chemical Industries, Ltd. are dissolved to a concentration of 1.4% (w / w) and 2.0% (w / w), respectively. The purified polypeptides obtained in Examples A-1 to A-4 were separately and uniformly mixed, and then adjusted to pH 7.2 to obtain a pasty product containing about 10 mg of the polypeptide per gram.
[0111]
Each product has excellent spreadability and stability, and is useful as an ointment for treating and preventing malignant tumors, acute leukemias, malignant lymphomas, chronic leukemias, and sensitive diseases including T-cell leukemia.
[0112]
Example B-5
<tablet>
The purified polypeptide obtained in Examples A-1 to A-4 and lumine as a cell activator were uniformly mixed in anhydrous crystal α-maltose powder “Finetose” manufactured by Hayashibara, and the resulting mixture was compressed with a tablet press. Tablets containing about 5 mg each of the polypeptide and lumine per tablet (about 200 mg) were obtained.
[0113]
Each product has excellent ingestion and stability, and also has cell activation. Tablets for treating and preventing malignant tumors, acute leukemias, malignant lymphomas, chronic leukemias, and sensitive diseases including T-cell leukemia Useful as.
[0114]
【The invention's effect】
This invention is based on the discovery of a polypeptide derived from a mammal having L-asparaginase activity. All of the polypeptides of the present invention are substances whose amino acid sequences have been elucidated, and have the activity of hydrolyzing stable L-asparagine. Thereby, the polypeptide of this invention exhibits power as a therapeutic agent / preventive agent for various diseases caused by L-asparagine-dependent tumor cells.
[0115]
Since the polypeptide of the present invention is derived from mammals, it has low antigenicity to humans and does not cause serious side effects even when administered in large doses or continuously. Therefore, the polypeptide of the present invention has an advantage that a desired effect can be exerted without strictly controlling the sensitivity of the patient at the time of use.
[0116]
Thus useful polypeptide of the present invention can be easily produced in a desired amount by utilizing the DNA of the present invention encoding the polypeptide.
[0117]
The present invention exhibits such remarkable operational effects, and it can be said that it is a very significant invention to contribute to the world.
[0118]
[Sequence Listing]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[Brief description of the drawings]
FIG. 1 is a diagram showing an overview of an overlap wrap extension method.
FIG. 2 is a diagram showing a restriction enzyme map of recombinant DNA pKGPA / WT.
FIG. 3 is a diagram showing an outline of a method for preparing recombinant DNA pBIgGPA / WT.
FIG. 4 is a diagram showing a restriction enzyme map of recombinant DNA pBIgGPA / WT.
FIG. 5 is a view showing a restriction enzyme map of recombinant DNA pKGPA / D364stp.
FIG. 6 is a diagram showing a restriction enzyme map of recombinant DNA pKHA / MUT1.
FIG. 7 is a diagram showing a restriction enzyme map of recombinant DNA pKHA / MUT2.
FIG. 8 is a diagram showing a restriction enzyme map of recombinant DNA pKHA / MUT3.
FIG. 9 is a diagram showing a restriction enzyme map of recombinant DNA pKHA / MUT5.
FIG. 10 shows a restriction enzyme map of recombinant DNA pBIgGPA / D364stp.
FIG. 11 is a diagram showing a restriction enzyme map of recombinant DNA pBIgHA / MUT1.
FIG. 12 is a diagram showing a restriction enzyme map of recombinant DNA pBIgHA / MUT2.
FIG. 13 is a diagram showing a restriction enzyme map of recombinant DNA pBIgHA / MUT3.
FIG. 14 is a diagram showing a restriction enzyme map of recombinant DNA pBIgHA / MUT5.
[Explanation of symbols]
Eco RI Eco RI cleavage site
Hin dIII Hin dIII cleavage site
Not I Not I cleavage site
Xho I Xho I cleavage site
GPA / WT DNA encoding guinea pig wild-type L-asparaginase (GPA / WT DNA)
D364stp DNA encoding guinea pig L-asparaginase homolog (GPA / D364stpDNA)
HA / MUT1 DNA encoding human L-asparaginase homologue (HA / MUT1 DNA)
HA / MUT2 DNA encoding human L-asparaginase homologue (HA / MUT2 DNA)
HA / MUT3 DNA encoding human L-asparaginase homologue (HA / MUT3 DNA)
HA / MUT5 DNA encoding human L-asparaginase homolog (HA / MUT5 DNA)
Ptac tac promoter
rrnBT1T2 Ribosomal RNA operon transcription termination region
5S 5S ribosomal RNA gene
AmpR ampicillin resistance gene
pBR322ori Origin of replication in E. coli
DNA encoding a peptide portion containing a secretory signal sequence of Ig sec immunoglobulin (Ig sec DNA)
Emsv Moloney Mouse Sarcoma Virus Long Terminal Repeat Enhancer
Promoter derived from Pmti mouse metallothionein I gene
Poly (A) SV40-derived poly (A) addition signal
BPVI Bovine papilloma virus genome

Claims (3)

A human L-asparaginase mutant polypeptide having an L-asparaginase activity comprising the amino acid sequence shown in any one of SEQ ID NOs: 6 to 9 in the sequence listing , or the mutant without substantially losing the activity A human L-asparaginase mutant polypeptide in which one or more amino acids of the polypeptide are substituted with other amino acids. A sensitive disease agent comprising the polypeptide according to claim 1 as an active ingredient. The sensitive disease agent according to claim 2 as a therapeutic agent for malignant tumor , leukemia or lymphoma .

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