JP3913316B2 - Method for removing N-terminal methionine - Google Patents

Description

［式（I）中、Ｘはアミノ酸残基または２以上の任意のアミノ酸数を有するペプチド鎖であればいずれでもよいが、実用的な面からは、遺伝子工学的に製造された蛋白質のＸに対応する部分のペプチド鎖が挙げられる。なお、本願明細書において、蛋白質あるいはペプチドと称する場合、複数のアミノ酸からなるペプチドまたは蛋白質は、非グリコシル化またはグリコシル化ペプチドまたは蛋白質のいずれであってもよい。］
【０００５】
すなわち、本発明は、
（１）Ｎ末端に酸化されていてもよいメチオニン残基を有するペプチドまたはその塩とα−ジケトン類を反応させた後、加水分解することを特徴とする該メチオニン残基の除去方法；
（２）Ｎ末端に酸化されていてもよいメチオニン残基を有するペプチドが遺伝子工学的に製造されたペプチドである前記（１）記載の方法；
（３）遺伝子工学的に製造されたペプチドがＮ末端に酸化されていてもよいメチオニン残基が付加した成長ホルモン、ニューロトロフィン−３、ベータセルリン、副甲状腺ホルモンまたはインターロイキン−２である前記（２）記載の方法；
（４）遷移金属イオンの存在下にα−ジケトン類を反応させることを特徴とする前記（１）記載の方法；
（５）塩基の存在下にα−ジケトン類を反応させることを特徴とする前記（１）記載の方法；
（６）遷移金属イオンおよび塩基の存在下にα−ジケトン類を反応させることを特徴とする前記（１）記載の方法；
（７）α−ジケトン類がグリオキシル酸またはその塩である前記（１）記載の方法；
（８）遷移金属イオンが銅イオンである前記（４）記載の方法；
（９）塩基がピリジンである前記（５）記載の方法；
（１０）塩基を用いて加水分解することを特徴とする前記（１）記載の方法；
（１１）塩基がアミン類である前記（１０）記載の方法；
（１２）塩基がジアミン類またはチオもしくはセレノセミカルバジド類である前記（１０）記載の方法；
（１３）ジアミン類がｏ−フェニレンジアミンである前記（１２）記載の方法；
（１４）遺伝子工学的に製造され、Ｎ末端にメチオニンが付加したヒト成長ホルモンまたはその塩とグリオキシル酸またはその塩とを硫酸銅およびピリジンの存在下に反応させた後、ｏ−フェニレンジアミンと反応させることを特徴とするヒト成長ホルモンまたはその塩の製造法；
（１５）遺伝子工学的に製造され、Ｎ末端にメチオニンが付加したニューロトロフィン−３またはその塩とグリオキシル酸またはその塩とを硫酸銅およびピリジンの存在下に反応させた後、ｏ−フェニレンジアミンと反応させることを特徴とするニューロトロフィン−３またはその塩の製造法；
（１６）遺伝子工学的に製造され、Ｎ末端にメチオニンが付加したヒトベータセルリンまたはその塩とグリオキシル酸またはその塩とを硫酸銅およびピリジンの存在下に反応させた後、ｏ−フェニレンジアミンと反応させることを特徴とするヒトベータセルリンまたはその塩の製造法；
（１７）遺伝子工学的に製造され、Ｎ末端にメチオニンが付加したヒトインターロイキン−２またはその塩とグリオキシル酸またはその塩とを硫酸銅およびピリジンの存在下に反応させた後、ｏ−フェニレンジアミンと反応させることを特徴とするヒトインターロイキン−２またはその塩の製造法；
（１８）遺伝子工学的に製造され、Ｎ末端にメチオニンが付加した副甲状腺ホルモンまたはその塩とグリオキシル酸またはその塩とを硫酸銅およびピリジンの存在下に反応させた後、ｏ−フェニレンジアミンと反応させることを特徴とする副甲状腺ホルモンまたはその塩の製造法；
（１９）式 CH₃-S(O)_m-(CH₂)₂-CO-CO-Ｘ［式中、ｍは０ないし２の整数を示し、Ｘはアミノ酸残基またはペプチド鎖を示す。］で表される化合物またはその塩；および
（２０）前記（１９）記載の化合物を加水分解することを特徴とするアミノ酸、ペプチドまたはその塩の製造法；などに関する。
【０００６】
本明細書において、酸化されていてもよいメチオニン残基は、メチオニン残基またはそのＳ酸化体を示し、メチオニン残基のＳ酸化体としては、スルホキシドおよびスルホン体が挙げられる。
Ｎ末端に酸化されていてもよいメチオニン残基を有するペプチドまたは蛋白質としては、式CH₃-S(O)_m-(CH₂)₂-CH(NH₂)-CO-Ｘ［式中、ｍは０ないし２の整数を示し、Ｘはアミノ酸残基またはペプチド鎖を示す。］で表されるペプチドまたは蛋白質が挙げられ、これらは塩を形成してもよく、塩としては、本発明の反応を阻害しないものであれば何れでもよいが、中でも薬学的に許容可能な塩が好ましく、塩酸、臭化水素酸、硝酸、硫酸、リン酸など無機酸との塩、酢酸、フタル酸、フマル酸、酒石酸、マレイン酸、クエン酸、コハク酸、メタンスルホン酸、ｐ−トルエンスルホン酸などの有機酸との塩、ナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩などのアルカリ土類金属塩、アンモニウム塩などがあげられる。
【０００７】
上記式中、ｍとしては０が好ましい。また、Ｘとしてはアミノ酸の数が２以上のペプチド鎖が好ましい。
本発明の除去方法に用いるペプチドとしては、アミノ酸数が５０未満のいわゆるペプチドあるいはアミノ酸数が５０以上のいわゆる蛋白質の何れであってもよい。
このように、本願明細書において、「ペプチド」で示される用語は、アミノ酸数が５０未満の分子のみならず、アミノ酸数が５０以上の分子をも含むものであるが、なかでも、アミノ酸数が５０以上の分子（いわゆる蛋白質）が好ましく用いられる。
好ましいペプチドとしては、アミノ酸数が２ないし１０００であるペプチド、さらに好ましくはアミノ酸数が１５ないし５００であるペプチドが挙げられ、その具体例としては、成長ホルモン（ＧＨ）、副甲状腺ホルモン（ＰＴＨ）、インシュリン、神経成長因子、脳由来神経栄養因子、毛様体神経栄養因子、グリア由来神経栄養因子、ニューロトロフィン−３、４または６、中枢神経成長因子、グリア成長因子、肺由来神経栄養因子、上皮細胞成長因子、繊維芽細胞成長因子、血小板由来成長因子、トランスフォーミング成長因子αまたはβ、血管内皮細胞成長因子、ティッシュ・プラスミノーゲン・アクチベータ、ウロキナーゼ、プロテインＣ、トロンボモジュリン、骨形成因子、カルシトニン、インスリン様成長因子、インターフェロン−α、βまたはγ、インターロイキン−１（α、β）〜１２、顆粒コロニー刺激因子、顆粒マクロファージ・コロニー刺激因子、顆粒マクロファージ刺激因子、トロンボポエチン、エリスロポイエチン、ＰＡＣＡＰ、心房性ナトリウム利尿ペプチド、エンドセリン、巨核球成長因子、血液幹細胞成長因子、肝細胞成長因子、モチリン、イムノトキシン、腫瘍壊死因子、ヒルジン、コルチコトロピン、アンジオテンシン、アンジオテンシン２およびそのペプチド性拮抗薬、アンジオテンシン３、ブラジキニン類、ブラジキニン増強因子、α、βまたはγエンドルフィン、エンケファリン、好球中走化性因子、ガストリン、グルカゴン、成長ホルモン放出因子、キョウトルフィン、カリジン、性腺刺激ホルモン放出ホルモン、肥満細胞脱顆粒ペプチド、メラニン細胞刺激ホルモン、ニューロテンシン、トリプシンインヒビター、オキシトシン、プロインシュリンＣ−ペプチド、セクレチン、ソマトスタチン、甲状腺刺激ホルモン放出ホルモン、ユビキチン、ウロガストロン、バソプレッシン類、キニン類、タフトシン、ソマトメジン、コルチコトロピン放出因子、インスリン様成長因子、カルシトニン遺伝子関連ペプチド、ＰＴＨrＰ、ＶＩＰ、ＤＨＩ、インスリノトロピン、ＧＲＰ、ＣＣＫ−ＰＺ、Galanin（ガラニン）、アカトラムペプチド（Antrum Peptide）、ＰＰＹ、Pancreatic Polypeptide、ＰＳＰ、パンクレアスタチン、hＣＧ、hＣＳ、リラキシン、血清胸腺因子、サイモポイエチン、サイモシン、ファクターXIII、ファクターVIII、プロウロキナーゼ、ＳＯＤ、ファクターVIIａ、アンチトロンビンなどの蛋白質およびそれらのムテイン（天然型の蛋白質に１つ以上のアミノ酸が置換、欠損または付加し、天然の蛋白質と同等またはそれ以上の生物学的または免疫学的活性を示すもの）など、あるいは化学合成などにより製造される公知または新規のペプチドなどが挙げられるが、なかでも、遺伝子工学的に製造された蛋白質、とりわけ、遺伝子工学的に製造され、Ｎ末端に酸化されていてもよいメチオニンが付加した成長ホルモン、ニューロトロフィン−３、ベータセルリン、副甲状腺ホルモン、インターロイキン−２などが好ましく用いられる。
上記した天然型の蛋白質は、何れの動物種由来のものであってもよいが、実用的には、ヒト由来の蛋白質が好ましく用いられる。
【０００８】
本明細書において、α−ジケトン類は、上記したペプチドまたはその塩のアミノ基移転反応を進行させうるものであれば何れでもよく、例えば式Ｒ¹−ＣＯ−ＣＯ−Ｒ²［式中、Ｒ¹は水素またはカルボキシル基で置換されていてもよい低級アルキルもしくはフェニル基（好ましくは水素またはメチル、さらに好ましくは水素）を示し、Ｒ²は水酸基、低級アルコキシ基または低級アルキルで置換されていてもよいアミノ基（好ましくは水酸基）を示す。］で表される化合物またはその塩などが挙げられる。上記式中、Ｒ¹で示される低級アルキル基としては、メチル、エチル、プロピル、ｉ−プロピル、ブチル、ｉ−ブチル、ｓｅｃ−ブチル、ｔ−ブチルなどの炭素数１ないし６程度のアルキル基などが挙げられ、Ｒ²で示される低級アルコキシ基としては、メトキシ、エトキシ、プロポキシ、ｉ−プロポキシ、ブトキシ、ｉ−ブトキシ、ｓｅｃ−ブトキシ、ｔ−ブトキシなどの炭素数１ないし６程度のアルコキシ基などが挙げられる。また、Ｒ²で示される低級アルキルで置換されていてもよいアミノ基としては、前記したＲ¹で示される低級アルキル基を１ないし２個有していてもよいアミノ基などが挙げられる。さらに、塩としては、上記したペプチドまたは蛋白質の塩と同様なものが挙げられる。α−ジケトン類の具体例としては、グリオキシル酸、ピルビン酸、オキサル酢酸、フェニルグリオキシル酸、２−オキソグルタル酸などが挙げられるが、なかでも、グリオキシル酸が好ましく用いられる。
【０００９】
Ｎ末端に酸化されていてもよいメチオニン残基を有するペプチドまたはその塩とα−ジケトン類とのアミノ基転移反応は、通常、ペプチドまたはその塩１モルに対して、１ないし１万モル（好ましくは２０００ないし４０００モル）程度のα−ジケトン類を、約０ないし７０℃（好ましくは約２０ないし４０℃）で約５分ないし２時間（好ましくは約１５分ないし１時間）反応させるのが好ましい。上記したアミノ基転移反応を阻害しないものであれば何れの緩衝液（例、リン酸緩衝液、酢酸緩衝液、クエン酸緩衝液など）を用いてもよいが、なかでも、酢酸緩衝液が好ましく用いられる。また、反応のｐＨは、約２ないし９、なかでも、約４ないし７、とりわけ、約５ないし６に調整して、Ｎ末端に酸化されていてもよいメチオニン残基を有するペプチドまたはその塩が変性しない条件下で反応を進行させるのがよい。
該アミノ基転移反応を促進するため、遷移金属イオンの存在下にα−ジケトン類を反応させることが好ましく、通常、α−ジケトン類１モルに対して、０.００１ないし０.１モル（好ましくは０.０１ないし０.０５モル）程度の遷移金属イオンを用いるのが好ましい。遷移金属イオンとしては、例えば、銅イオン（Cu⁺，Cu²⁺）、コバルトイオン（Co²⁺，Co³⁺）、ニッケルイオン（Ni²⁺，Ni³⁺）、鉄イオン（Fe²⁺，Fe³⁺）、亜鉛イオン（Zn²⁺）、アルミニウムイオン（Al³⁺）、マンガンイオン（Mn²⁺など）、ガリウムイオン（Ga³⁺）、インジウムイオン（In³⁺）、マグネシウムイオン（Mg²⁺）、カルシウムイオン（Ca²⁺）などを用いることができるが、なかでも、銅イオン、コバルトイオンなど、とりわけ、銅イオン（Cu²⁺）が好ましく用いられる。これらの遷移金属イオンは、通常、硫酸、硝酸、塩酸、過塩素酸などの無機酸との塩または酢酸、シュウ酸、クエン酸、炭酸などの有機酸との塩として、反応溶媒に添加することができ、なかでも、硫酸銅、酢酸銅、とりわけ、硫酸銅が好ましく用いられる。
【００１０】
また、塩基の存在下にα−ジケトン類を反応させることが好ましく、通常、α−ジケトン類１モルに対して、０.１ないし２モル（好ましくは０.５ないし１.０モル）程度の塩基を用いるのが好ましい。塩基としては、例えば、トリエチルアミン、トリブチルアミンなどのアルキルアミン類、Ｎ，Ｎ−ジメチルアニリン、ピリジン、ルチジン、コリジン、４−（ジメチルアミノ）ピリジン、イミダゾールなどの芳香族アミン類、尿素などの有機塩基などを用いることができるが、なかでも、芳香族アミン類、とりわけ、ピリジンが好ましく用いられる。
【００１１】
さらに、上記したアミノ基転移反応は、遷移金属イオンおよび塩基の存在下にα−ジケトン類を反応させることが好ましく、実用的には、遷移金属イオン、塩基およびα−ジケトン類の３成分（例えば、硫酸銅、ピリジンおよびグリオキシル酸など）を含有する混合液を、Ｎ末端に酸化されていてもよいメチオニン残基を有するペプチドまたはその塩を含有する水溶液に添加して、アミノ基転移反応を進行させる。
該アミノ基転移反応により得られ、式 CH₃-S(O)_m-(CH₂)₂-CO-CO-Ｘ［式中、ｍは０ないし２の整数を示し、Ｘはアミノ酸残基またはペプチド鎖を示す。]で表される化合物またはその塩は、新規な化合物であり、ペプチドまたは蛋白質の公知精製手段、例えば、抽出、塩析、分配、再結晶、クロマトグラフィーなどにより、反応溶液から単離・精製することもできるが、そのまま次の加水分解反応に付すこともできる。
アミノ基転移反応で得られたジケトン体は、通常、塩基による加水分解反応に付して、Ｎ末端の酸化されていてもよいメチオニン残基が除去されたアミノ酸、ペプチドまたはその塩に変換することができる。
【００１２】
加水分解反応に用いる塩基としては、例えば、システアミン、トリエチルアミン、トリブチルアミンなどのアルキルアミン類、Ｎ，Ｎ−ジメチルアニリン、ピリジン、ルチジン、コリジン、４−（ジメチルアミノ）ピリジン、イミダゾールなどの芳香族アミン類、ｏ−フェニレンジアミン、トリレン−３，４−ジアミン、３,４−ジアミノ安息香酸、２,３−ジアミノフェノール、４−クロロ−ｏ−フェニレンジアミンなどのジアミン類（好ましくは芳香族ジアミン類、なかでも、ｏ−フェニレンジアミン類）、チオセミカルバジド、アセトンチオセミカルバジド、フェニルチオセミカルバジドなどのチオセミカルバジド類、セレノセミカルバジド、アセトンセレノセミカルバジドなどのセレノセミカルバジド類などのアミン類などを用いることができるが、なかでも、アミン類、とりわけ、ジアミン類またはチオセミカルバジド類が好ましく用いられ、特に、ｏ−フェニレンジアミンが好ましく用いられる。
【００１３】
塩基の量は、通常、ジケトン体１モルに対して約１ないし１万モル、好ましくは約５００ないし２０００モルである。加水分解反応は、通常、約０ないし７０℃（好ましくは約２０ないし４０℃）で約１ないし５０時間（好ましくは約１０ないし２５時間）で進行させるのが好ましい。反応には、緩衝液を溶媒として用いることが好ましく、緩衝液としては、リン酸緩衝液、酢酸緩衝液、クエン酸緩衝液などが挙げられる。上記した加水分解反応を阻害しないものであれば何れの緩衝液を用いてもよいが、なかでも、酢酸緩衝液が好ましく用いられる。また、反応のｐＨは、約２ないし９、なかでも、約３ないし７、とりわけ、約４ないし６の中性付近に調整して、得られるアミノ酸、ペプチドまたはその塩が変性しない条件下で反応を進行させるのがよい。
このようにして得られるアミノ酸、ペプチドまたはその塩は、公知の精製手段、例えば、抽出、塩析、分配、再結晶、クロマトグラフィーなどにより、反応溶液から単離・精製することもできるが、好ましい例として、例えば、ＳＰ−セファロース（ファルマシア バイオテク(株)）あるいは、ＤＥＡＥ−５ＰＷ（トーソー(株)）を介したイオン交換クロマトグラフィーなどによる精製法が挙げられる。
【００１４】
本発明により製造されるポリペプチドはそのＮ末端にメチオニンを有さず、また天然の生理活性ポリペプチドと同一のアミノ酸配列を有するものとして得られるので、天然のポリペプチドと同様の活性を有し低毒性で安全に医薬品や診断用薬剤として使用できる。
本発明により、メチオニンの付加したペプチドからＮ末端メチオニンを特異的に除去することができる。
本発明の明細書および〔図面〕においてアミノ酸等の略号で表示する場合には、ＩＵＰＡＣ−ＩＵＢ Commission on Biochemical Nomenclature による略号あるいは当該分野における慣用略号に基づくものであり、その例を下に示す。またアミノ酸に関して光学異性がある場合は、特に明示しなければＬ−体を示す。
【００１５】
ＳＤＳ ： ドデシル硫酸ナトリウム
Ｇly ： グリシン
Ａla ： アラニン
Ｖal ： バリン
Ｌeu ： ロイシン
Ｉle ： イソロイシン
Ｓer ： セリン
Ｔhr ： スレオニン
Ｃys ： システイン
Ｍet ： メチオニン
Ｇlu ： グルタミン酸
Ｇln ： グルタミン
Ａsp ： アスパラギン酸
Ａsn ： アスパラギン
Ｌys ： リジン
Ａrg ： アルギニン
Ｈis ： ヒスチジン
Ｐhe ： フェニルアラニン
Ｔyr ： チロシン
Ｔrp ： トリプトファン
Ｐro ： プロリン
Ａsx ： Ａsp ＋ Ａsn
Ｇlx ： Ｇlu ＋ Ｇln
【００１６】
【発明の実施の形態】
以下の参考例および実施例によって本発明をより具体的に説明するが、本発明はこれらに制限されるものではない。
【実施例】
参考例１
Ｍet−rhＧＨの製造：
特開昭６２−１７１６９９の参考例４に記載の方法に準じて、Ｍet−rhＧＨを製造した。
（i） 生産菌
ＡＴＣＣより分譲されたヒト成長ホルモン遺伝子を有する形質転換体エシェリヒア・コリ Ｋ１２ χ１７７６／pＨＧＨ １０７（ＡＴＣＣ ３１５３８）を使用した。
（ii）培 養
形質転換体エシェリヒア・コリ Ｋ１２ χ１７７６／pＨＧＨ １０７（ＡＴＣＣ ３１５３８，ＩＦＯ １４５０５）を２L 容マイヤーのバクト・トリプトン１％，バクト・イースト０.５％，食塩０.５％，テトラサイクリン・塩酸１０mg／L，アンピシリン・ナトリウム１０mg／L，チミン２０mg／L およびジアミノピメリン酸１００mg／L を含む液体培地（pＨ ７.０）１L に接種して３７℃で一晩回転振盪培養した。この培養液を、リン酸１水素ナトリウム１.６８％，リン酸２水素カリウム０.３０％，塩化アンモニウム０.１０％，食塩０.０５％，消泡剤２００mg／L，グルコース１.００％，カザミノ酸１.００％，硫酸マグネシウム２４６mg／L，テトラサイクリン・塩酸１０mg／L，アンピシリン・ナトリウム１０mg／L，チミン２０mg／L およびジアミノピメリン酸１００mg／L を含む液体培地（pＨ ６.８）２０L の入った５０L 容ジャーファーメンターに移し、３７℃で１０時間通気撹拌培養した。この培養液を遠心分離し、菌体を集めた。
【００１７】
参考例２
参考例１で得た湿菌体２kgを取り出し、この菌体に８Ｍ塩酸グアニジンを含む５０mＭトリス塩酸緩衝液（pＨ ８.０）６L を加え、菌体の固まりがなくなるまで十分撹拌し菌体を溶解した後、遠心分離（１０,０００rpm、６０分）を行い約６L の菌体抽出液を得た。この菌体抽出液を約７０L の１０mＭトリス塩酸緩衝液（pＨ ７.０）に対して２回透析し、透析終了後得られた透析内液約９L に、最終濃度が２０％飽和硫酸アンモニウム濃度になるように硫酸アンモニウムを加えて溶解した後、遠心分離（４,２００rpm、６０分）を行い約１０L の遠心上澄液を得た。この上澄液を２回に分けてフェニル−トヨパール６５０Ｃカラム（５cmφ×５０cm）に通液し、吸着、洗浄した後、▲１▼４０％飽和硫酸アンモニウムを含む１０mＭトリス塩酸緩衝液（pＨ ７.０）と▲２▼１０mＭトリス塩酸緩衝液（pＨ７.０）とによる濃度勾配溶出法により溶出し、Ｍet−rhＧＨ画分を集め、５０mＭ炭酸水素ナトリウム溶液（pＨ ８.２）に対して透析した。得られた透析内液を遠心分離（４,２００rpm、４５分）し、約３.７L の遠心上澄液を得た。この上澄液を４回に分けてＤＥＡＥ−トヨパール６５０Ｍカラム（４cmφ×５０cm）に通液し、吸着、洗浄した後、▲１▼１０mＭ炭酸水素ナトリウム溶液（pＨ ８.２）と▲２▼１Ｍ塩化ナトリウムを含む１０mＭ炭酸水素ナトリウム溶液（pＨ ８.２）とによる濃度勾配溶出法によりＭet−rhＧＨを溶出し、集めたＭet−rhＧＨ画分を５０mＭ炭酸水素ナトリウム溶液（pＨ ８.２）に対して透析した。得られた約１.２L の透析内液をＤＥＡＥ−５ＰＷカラム（５５mmφ×２０cm、東ソー）及び▲１▼１０mＭ炭酸水素ナトリウム溶液（pＨ ８.２）と▲２▼１Ｍ塩化ナトリウムを含む１０mＭ炭酸水素ナトリウム溶液（pＨ ８.２）とによる濃度勾配溶出法を用いた高速液体クロマトグラフィーにより吸着、溶出し、約６００mlのＭet−rhＧＨ画分を得た。この溶液をアミコンダイアフロー（ＹＭ−１０膜、７６mmφ、アミコン社）で濃縮した後、この濃縮液約２９４mlを、１００mＭ炭酸水素ナトリウム溶液（pＨ ８.２）で平衡化したトヨパールＨＷ−５０Ｆカラム（８cmφ×５０cm）を用いてゲルろ過を行い、Ｍet−rhＧＨ画分を得た。ついでマイレクスＧＶフィルター（０.２２μm、ミリポア社）でろ過し、８６９mgのＭet−rhＧＨを得た。
【００１８】
実施例１
参考例２で得られたＮ末端にメチオニンの付加したヒト成長ホルモン（Ｍet−rhＧＨ）５０mgを凍結乾燥した後、５０mＭリン酸緩衝液（pＨ ８.０）４０mlに溶解した後、０.１Ｍ硫酸銅５ml、グリオキシル酸２.３ｇ、ピリジン５mlの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２Ｍ酢酸−２Ｍ酢酸ナトリウム溶液で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５mmID×６００mmL）に通液し、平衡化に用いた溶液を６ml／分の流速で展開し、Ｍet−rhＧＨのジケトン体画分をプールした。続いてこの画分に、ｏ−フェニレンジアミンを２０mＭ濃度になるように添加して、脱気、窒素ガスシールを行った後、３７℃で２０時間反応した。反応終了後、反応液を２０mＭトリス塩酸緩衝液（pＨ ８.０）で平衡化したセファデックスＧ−２５カラム（２５mmID×６００mmL）に通液し、平衡化に用いた緩衝液を６ml／分の流速で展開し、Ｎ末端にメチオニンの付加していないヒト成長ホルモン（rhＧＨ）画分をプールした。プールしたrhＧＨ画分を２０mＭトリス塩酸緩衝液（pＨ ８.０）で平衡化したＤＥＡＥ−５ＰＷ（２１.５mmID×１５０mmL）に吸着した後、０〜１００％Ｂ（Ｂ＝２０mＭトリス塩酸緩衝液＋１Ｍ ＮaＣl、pＨ８.０）の段階勾配で３０分間、８.５ml／分の流速で溶出を行い、rhＧＨ画分をプールした。さらに、５％エタノールで平衡化したトヨパールＨＷ−５０（２０mmID×６００mmL）（トーソー(株)）にrhＧＨ画分を通液した後、平衡化に用いた溶液を６ml／分の流速で展開し、rhＧＨ画分をプールした後、凍結乾燥を行い、rhＧＨの凍結乾燥粉末を得た。
【００１９】
実施例２
（rhＧＨの特徴決定）
ａ) ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例１で得られたrhＧＨを Sample buffer［Laemmli, ネイチャー（Nature），２２７，６８０（１９７０）］に懸濁し、１００mＭ ＤＴＴにより１００℃で１分間加熱した後、マルチゲル１０／２０（第一化学薬品(株)）で電気泳動を行った。泳動後のゲルをクマシーブリリアントブルー（Coomassie brilliant blue）で染色したところ、単一バンドの蛋白質が認められ、精製品はほぼ単一であった。結果を〔図１〕に示す。〔図１〕において、Lane １〜３はrhＧＨ（３μg）、blank および分子量マーカーを示す。
ｂ) Ｎ末端アミノ酸配列分析
実施例１で得られたrhＧＨのＮ末端アミノ酸配列を、気相プロテインシーケンサー（アプライド バイオシステムズ モデル４７７Ａ）を用いて決定した。その結果、得られたrhＧＨのＮ末端アミノ酸配列は cＤＮＡの塩基配列から推定したrhＧＨのＮ末端アミノ酸配列と一致した。結果を〔表１〕に示す。
【表１】

【００２０】
ｃ) アミノ酸組成分析
実施例１で得られたrhＧＨを約２０μg 用い、そのアミノ酸組成をアミノ酸分析計（ベックマン システム６３００Ｅ）により決定した。その結果、rhＧＨの cＤＮＡの塩基配列から推定したアミノ酸組成と一致した。結果を〔表２〕に示す。
【表２】

ｄ) Ｃ末端アミノ酸分析
実施例１で得られたrhＧＨを１５n mol用いて、そのＣ末端アミノ酸をアミノ酸分析計（ベックマン システム６３００Ｅ）により決定した。rhＧＨの cＤＮＡの塩基配列から推定したＣ末端アミノ酸と一致した。結果を〔表３〕に示す。
【表３】

【００２１】
実施例３
（rhＧＨの活性測定）
Ｎb ２細胞を用いる「ジャーナル オブ クリニカル エンドクリノロジー アンド メタボリズム（Journal of Clinical Endocrinology ＆ Metabolism），５１，１０５８（１９８０）」に記載の方法で測定したところ、実施例１で得られたrhＧＨの精製品は、標準品とほぼ同等の活性を有していた。
【００２２】
参考例３
Ｍet−ＮＴ−３の製造：
特願平８−７４７７５号明細書の参考例１〜３および実施例１〜２に記載の方法に準じてＭet−ＮＴ−３を製造した。
（１）ＮＴ−３ ＤＮＡのクローニング
E. coli Ｙ１０９０にヒトグリオーマ由来のλgt １１ cＤＮＡライブラリー（Clontech Laboratories, Inc.）を感染させたのち、約６×１０⁵個のファージをＮＺＣＹ培地（Molecular Cloning, A Laboratory Manual, Clod Spring Harbor Laboratory, １９８２に記載）にまき、３７℃で５時間培養した。次にナイロン膜をプレート上にのせ、１分間放置後、プレートからはずした。このナイロン膜を０.５Ｍ ＮaＯＨ−１.５Ｍ ＮaＣl、ついで１.５Ｍ ＮaＣl−０.５Ｍ Tris−ＨＣl pＨ８.０に浸し、さらに２×ＳＳＣ〔Molecular Cloning, A Laboratory Manual 前掲参照〕に浸し、風乾後、８０℃で２時間放置した。
ヒトβＮＧＦ〔ネイチャー（Nature），３０３，８２１（１９８３）〕をコードするＤＮＡ（約０.３８kb）を化学合成し、ニックトランスレーションによって〔α−³²Ｐ〕dＣＴＰでラベル化することによってプローブを作製した。
上記で得られたナイロン膜とプローブを用いて Molecular Cloning, A Laboratory Manual, Clod Spring Harbor Laboratory, １９８２に記載の方法に従ってハイブリダイゼーションを行った。即ち、プローブを含むハイブリダイゼーション溶液にナイロン膜を浸し、６５℃で１６時間保温した。該ナイロン膜を室温において２×ＳＳＣ−０.１％ＳＤＳで洗浄したのち、６０℃において１×ＳＳＣ−０.１％ＳＤＳで洗浄した。次にオートラジオグラフィーによって陽性クローンを得た。このようにして得られたクローンλβＧＮ１３２１からＥcoＲＩで cＤＮＡを切り出し、プラスミド pＵＣ１１８（宝酒造株式会社製）のＥcoＲＩ部位に挿入し、プラスミド pＵＮＫ５を得た。
【００２３】
（２）大腸菌用のＮＴ−３発現ベクターの構築
参考例３（１）で得られたプラスミド pＵＮＫ５に挿入されているＮＴ−３ cＤＮＡには、ＮＴ−３のＮ末端の１１番目のチロシン残基をコードする領域付近にＳcaＩ部位が、ＮＴ−３の終止コドンの５０塩基下流付近にＮsiＩ部位が存在する。そこで pＵＮＫ５より０.３kb ＳcaＩ−ＮsiＩ断片を単離し、これにアダプターＮＧＦＴＥ−１（３５mer）、ＮＧＦＴＥ−２（３３mer）、ＮＧＦＴＥ−３（７mer）、ＮＧＦＴＥ−４（１５mer）をＴ４ＤＮＡリガーゼで連結したのち制限酵素ＮdeＩとＢamＨＩで処理し、０.３kb ＮdeＩ−ＢamＨＩ断片を得た。
該アダプターを次に示す。
ＮＧＦＴＥ−１：５’TATGTACGCGGAGCATAAGAGTCACCGAGGGGAGT 3’ ３５mer（配列番号：１）
ＮＧＦＴＥ−２：５’ACTCCCCTCGGTGACTCTTATGCTCCGCGTACA 3' ３３mer（配列番号：２）
ＮＧＦＴＥ−３：５’TGCCAGG 3' ７mer
ＮＧＦＴＥ−４：５’GATCCCTGGCATGCA 3' １５mer（配列番号：３）
一方、Ｔ７プロモーターを有する発現ベクター pＥＴ−３Ｃ〔Rosenberg et al., ジーン（Gene），５６，１２５（１９８７）〕をＮdeＩとＢamＨＩで切断し、４.４kb ＮdeＩ−ＢamＨＩ断片を単離した。
上記で得られた４.４kb ＮdeＩ−ＢamＨＩ断片と０.３kb ＮdeＩ−ＢamＨＩ断片をＴ４ＤＮＡリガーゼで連結したのち、Escherichia coli ＤＨ１に導入し、得られたアンピシリン耐性の形質転換体〔Escherichia coli ＤＨ１／pＥＮＧＦＴ１０３〕から単離したプラスミドを pＥＮＧＦＴ１０３と命名した。
【００２４】
（３）大腸菌用のＭet−ＮＴ−３の生産
参考例３（２）で得られたＮＴ−３発現ベクター pＥＮＧＦＴ１０３及びT7リゾチーム発現ベクター pＬysＳを用いて、Escherichia coli ＭＭ２９４（ＤＥ３）〔Molecular Endocrinology, ４，８６９（１９９０）〕の形質転換を行い、形質転換体 E. coli ＭＭ２９４（ＤＥ３）／pＬysＳ，pＥＮＧＦＴ１０３（ＩＦＯ １５９３２，ＦＥＲＭ ＢＰ−５４８３）を得た。
形質転換体 E. coli ＭＭ２９４（ＤＥ３）／pＬysＳ，pＥＮＧＦＴ１０３を５０μg／mlのアンピシリンと１５μg／mlのクロラムフェニコールを含むＬＢ培地〔１％ペプトン、０.５％酵母エキス、０.５％塩化ナトリウム〕１リットルを含む２リットル容フラスコで３０℃、８時間振とう培養した。得られた培養液を２０リットルの主醗酵培地〔１.６８％リン酸一水素ナトリウム、０.３％リン酸二水素カリウム、０.１％塩化アンモニウム、０.０５％塩化ナトリウム、０.０５％硫酸マグネシウム、０.０２％消泡剤、０.０００２５％硫酸第一鉄、０.０００５％塩酸チアミン、１.５％ブドウ糖、１.５％カザミノ酸〕を仕込んだ５０リットル容醗酵槽へ移植して、３０℃で通気撹拌培養を開始した。培養液の濁度が約５００クレット単位になった時点で、１００mg／L 分のイソプロピル−β−Ｄ−チオガラクトピラノシド（ＩＰＴＧ）を添加し、さらに培養を続け、７時間後に培養を終了した。この培養終了液を遠心分離して、約３４０ｇの湿菌体を得、−８０℃に凍結保存した。
【００２５】
（４）大腸菌用のＭet−ＮＴ−３の生産（大量生産）
上記形質転換体 E. coli ＭＭ２９４（ＤＥ３）／pＬysＳ，pＥＮＧＦＴ１０３を５０μg／mlのアンピシリンと１５μg／mlのクロラムフェニコールを含むＬＢ培地〔１％ペプトン、０.５％酵母エキス、０.５％塩化ナトリウム〕１リットルを含む２リットル容フラスコで３０℃、１６.５時間振とう培養した。得られた培養液を２０リットルのＬＢ培地〔０.０２％消泡剤、５０μg／mlのアンピシリン及び１５μg／mlのクロラムフェニコールを含む〕を仕込んだ５０リットル容醗酵槽へ移植して、３０℃、７時間通気撹拌培養した。この培養液を３６０リットルの主醗酵培地〔１.６８％リン酸一水素ナトリウム、０.３％リン酸二水素カリウム、０.１％塩化アンモニウム、０.０５％塩化ナトリウム、０.０５％硫酸マグネシウム、０.０２％消泡剤、０.０００２５％硫酸第一鉄、０.０００５％塩酸チアミン、１.５％ブドウ糖、１.５％カザミノ酸〕を仕込んだ５００リットル容醗酵槽へ移植して、３０℃で通気撹拌培養を開始した。培養液の濁度が約５００クレット単位になった時点で、１００mg／L 分のイソプロピル−β−Ｄ−チオガラクトピラノシド（ＩＰＴＧ）を添加し、さらに培養を続け、５.５時間後に培養を遠心分離して、約６kgの湿菌体を得、−８０℃に凍結保存した。
【００２６】
（５）Ｍet−ＮＴ−３の活性化
参考例３（３）で得た湿菌体のうち４０ｇを取り出し、この菌体に１０mＭ ＥＤＴＡ（pＨ７.０）２４０mlを加えて懸濁した後、氷冷下でソニファイアー４５０（ブランソン社）を使って超音波により細胞を破砕し、遠心分離（１００００rpm、１時間）を行った。得られたペレットを同様の操作を２回行って洗浄した。次いで、５０mＭトリス塩酸／４Ｍ尿素／５mＭジチオスレイトール（ＤＴＴ）（pＨ８.０）を１６０ml加えてホモジナイズした後遠心分離を行い、得られたペレットに２０mＭクエン酸／８Ｍ尿素（pＨ３.０）を１２０mＭ加えて溶解後、遠心分離を行い、上澄液と沈降物を分離し、沈降物に対して再度同様の操作を行い得られた上澄液と先に得られた上澄液を混合し、２４０mlのペレット溶解液を得た。
この溶解液に１００mＭ酢酸溶液を７６０ml加えて希釈し、同じく１００mＭ酢酸で平衡化したセファデックスＧ−２５カラム（１１.３cmφ×５０）に通液し、尿素を除去した変性Ｍet−ＮＴ−３溶液１６４０mlを得た。この溶液を４℃で２日間静置した後、５０mＭリン酸緩衝液／１２.５％ショ糖（pＨ６.８）を加えて８.５L とし、５Ｍ水酸化ナトリウム又は濃リン酸を用いてpＨ６.０に調整し、再び４℃で２日間静置し活性化を行った。静置後２００mＭ硫酸銅を最終濃度が１０μＭになるように加え、撹拌後、さらに活性化を継続するため、再度４℃に２日間静置した。
【００２７】
（６）Ｍet−ＮＴ−３の精製
参考例３（５）で活性化を終了した溶液を、１００mＭリン酸緩衝液／０.１％３−［（３−コールアミドプロピル）ジメチルアンモニオ］−１−プロパンスルホネート（ＣＨＡＰＳ）（pＨ６.０）で平衡化したＳＰ−セファロース Fast Flowカラム（２.５cmφ×１２cm、ファルマシア バイオテク社）に通液し、吸着後２００mlの１００mＭリン酸緩衝液／０.１％ＣＨＡＰＳ／２００mＭ塩化ナトリウム（pＨ６.０）でカラムを洗浄し、次いで１００mＭリン酸緩衝液／０.１％ＣＨＡＰＳ／４００mＭ塩化ナトリウム（pＨ６.０）で溶出し、Ｍet−ＮＴ−３を含む溶出液を得た。この溶出液を Resource １５ＲＰＣカラム（２cmφ×３０cm、ファルマシア バイオテク社）に吸着した後、▲１▼１６％アセトニトリル／０.１％ＴＦＡと▲２▼３６％アセトニトリル／０.１％ＴＦＡによる濃度勾配溶出法で溶出し、その溶出液を凍結乾燥し、約２８mgのＭet−ＮＴ−３の白色粉末を得た。
【００２８】
実施例４
参考例３（６）で得られたＭet−ＮＴ−３ ５０mgを４.６mlの蒸留水で溶かした溶液に、グリオキシル酸９２.５mg、ピリジン２００μl、１００mＭ硫酸銅溶液２００μl を混合した溶液を加え、静かに撹拌した後、室温で１５分間静置し反応させた。反応終了後、この反応液を２Ｍ酢酸緩衝液（pＨ４.９）で平衡化したセファデックスＧ−２５カラム（２.５cmφ×５９cm）に通液し、Ｍet−ＮＴ−３のジケトン体３６mlを集めた。この溶液にｏ−フェニレンジアミン７８mgを加えて、３７℃で１５時間反応させた後、この反応終了液を、２Ｍ酢酸緩衝液（pＨ４.９）で平衡化したセファデックスＧ−２５カラム（２.５cmφ×５９cm）に通液し、得られたＮＴ−３画分７５mlを、ＯＤＰ−５０カラムを用いた▲１▼０.１％ＴＦＡと▲２▼０.１％ＴＦＡ／８０％アセトニトリルによる濃度勾配溶出法により、ＨＰＬＣで精製した後、得られたＮＴ−３画分を凍結乾燥し、ＮＴ−３の凍結乾燥粉末を得た。
【００２９】
実施例５
（ＮＴ−３の特徴決定）
ａ) ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例４で得られたＮＴ−３を Sample buffer［Laemmli, ネイチャー（Nature），２２７，６８０（１９７０）］に懸濁し、１００mＭ ＤＴＴにより１００℃で１分間加熱した後、マルチゲル１０／２０（第一化学薬品(株)）で電気泳動を行った。泳動後のゲルをクーマシーブリリアントブルーで染色したところ、単一バンドの蛋白が認められ、精製品はほぼ単一であった〔図２〕。〔図２〕において、Lane １〜２は分子量マーカーおよびＮＴ−３を示す。
【００３０】
ｂ) Ｎ末端アミノ酸配列分析
実施例４で得られたＮＴ−３のＮ末端アミノ酸配列分析を、気相プロテインシークェンサー４７７Ａ（モデル４７７Ａ、アプライドバイオシステムズ社）を用いて行った。その結果、得られたＮＴ−３のＮ末端アミノ酸配列は cＤＮＡの塩基配列から推定したＮＴ−３のＮ末端アミノ酸配列と一致した。結果を〔表４〕に示す。
【表４】

【００３１】
ｃ) アミノ酸組成分析
実施例４で得られたＮＴ−３を約２０μg 用いて、そのアミノ酸組成をアミノ酸分析計（システム６３００Ｅ、ベックマン社）により決定した。その結果、ＮＴ−３の cＤＮＡの塩基配列から推定したアミノ酸組成とよく一致した。結果を〔表５〕に示す。
【表５】

【００３２】
ｄ) Ｃ末端アミノ酸分析
実施例４で得られたＮＴ−３を１５n mol用いて、そのＣ末端アミノ酸をアミノ酸分析計（システム６３００Ｅ、ベックマン社）により決定した。その結果、ＮＴ−３の cＤＮＡの塩基配列から推定したＣ末端アミノ酸と一致した。結果を〔表６〕に示す。
【表６】

【００３３】
参考例４：Ｍet−ヒトＢＴＣの製造
特開平６−８７８９４の実施例４〜６、８および１３に記載の方法に準じて、Ｍet−ヒトＢＴＣを製造した。
（１）大腸菌のヒトＢＴＣ cＤＮＡ発現プラスミドの構築
成熟ヒトＢＴＣ（１−１４７アミノ酸残基）をコードする０.６Ｋb のＥcoＲＩ−ＢamＨＩ断片を、特開平６−８７８９４の実施例５に記載のプラスミド pＴＢ１５１５から単離した。ＡＴＧ翻訳開始コドン（配列番号４：５’ＴＡＴＧＧＡＴＧＧＧ ３’；配列番号５：５’ＡＡＴＴＣＣＣＡＴＣＣＡ ３’）を有する合成アダプターを上記０.６Ｋb 断片のＥcoＲＩ部位に連結した後、生成した０.６Ｋb ＮdeＩ−ＢamＨＩ断片を、Ｔ７プロモータ（ジーン、５６、１２５（１９８７））を含有するプラスミド pＥＴ−３ｃ中へ挿入し、プラスミド pＴＢ1505を構築した。
ヒトＢＴＣの８０のアミノ酸残基（特開平６−８７８９４の〔図１０−１〕〜〔図１０−２〕の１（Ａsp）から８０（Ｔyr）まで）をコードするＤＮＡ断片を得るため、鋳型としてプラスミド pＴＢ１５０５、プライマーとして２個のオリゴヌクレオチド（配列番号６：５’ＡＴＡＣＡＴＡＴＧＧＡＴＧＧＧＡＡＴＴＣＣＡ ３’；配列番号７：５’ＣＣＧＧＡＴＣＣＴＡＧＴＡＡＡＡＣＡＡＧＴＣＡＡＣＴＣＴ ３’）を用いてＰＣＲを行った。生成物をＮdeＩ及びＢamＨＩで消化し、２.０％アガロースゲル電気泳動で分画し、目的とする０.２５Ｋb ＤＮＡ断片を単離した。この０.２５Ｋb ＮdeＩ−ＢamＨＩ断片を、pＥＴ−３ｃのＴ７プロモータの下流にＴ４ＤＮＡリガーゼで連結挿入しプラスミド pＴＢ１５１６を得た（特開平６−８７８９４の〔図１３〕参照）。
【００３４】
（２）大腸菌中でのヒトＢＴＣの発現
E. coli ＭＭ２９４を、Ｔ７ファージのＲＮＡポリメラーゼ遺伝子で組換えられているラムダファージ（スチュディエ、スプラ）で溶原化した。その後、このプラスミド pＬysＳを E. coli ＭＭ２９４（ＤＥ３）へ導入し、E. coli ＭＭ２９４（ＤＥ３）／pＬysＳを得た。この菌体に参考例４（１）で得られたプラスミド pＴＢ１５１６を導入し、これにより、E. coli ＭＭ２９４（ＤＥ３）／pＬysＳ，pＴＢ１５１６を得た。
この形質転換細胞 E. coli ＭＭ２９４（ＤＥ３）／pＬysＳ，pＴＢ１５１６は、受託番号ＦＥＲＭ ＢＰ−３８３６で１９９２年４月２１日付で通産省工業技術院微生物工業研究所に寄託され、またこのものは１９９２年４月１６日付で受託番号ＩＦＯ １５２８２としてＩＦＯに寄託されている。
【００３５】
（３）組換え型大腸菌より産生されたＢＴＣの精製
参考例４（２）で得られた E. coli ＭＭ２９４（ＤＥ３）／pＬysＳ，pＴＢ１５１６を一晩培養した後、培養菌液を、２０倍希釈になるようにＬＢ培地に植菌した。３７℃で２時間培養後、ＩＰＴＧを最終濃度０.１mＭになるように添加し、さらに３時間培養した。遠心により菌体を集め使用時まで−２０℃に保存した。
５リットル培養相当の菌体ストックを解凍し、氷冷した５０mＭ Tris・ＨＣl（pＨ７.４），１０mＭ ＥＤＴＡ−０.２Ｍ ＮaＣl，１０％ sucrose, １mＭＡＰＭＳＦを含むバッファー３００mlに懸濁した。これに卵白リゾチーム４０mgを溶解し、４℃で２時間インキュベートした後、超音波処理を行い、２０,０００×ｇで１時間遠心して上清を取得した。この上清を２００mlのＱ−セファロースベットに通過させた後、ＴＣＡを最終濃度４％になるように加え、４０℃で１０分間静置した。２０,０００×ｇで２０分遠心して集めた沈殿を、１００mlの２０mＭ Tris（pＨ７.４），１mＭ ＥＤＴＡ−０.１５Ｍ ＮaＣl，１mＭ ＡＰＭＳＦを含むバッファーに懸濁し、乳鉢中でホモゲナイズしながら、５Ｍ ＮaＯＨを加え、pＨ６に調整した。このホモゲネートを１００,０００×ｇで１時間遠心して、得られた上清をＳ−Sepharose カラム（径１.６×１０cm，pharmacia）にかけた。カラムを０.１Ｍ potassium phosphate（pＨ６.０），１mＭ ＥＤＴＡ，０.５mＭ ＰＭＳＦを含むバッファーで洗浄した後、０Ｍから１ＭのＮaＣl 濃度勾配を４００ml，２００分にわたってかけ、溶出液を５ml毎に集めた。高い生物活性の認められた分画番号２０から２７を E. coli ＢＴＣ画分として、プールした。
E. coli ＢＴＣ画分にＴＦＡを最終濃度０.１％になるように添加し、Ｃ１８逆相ＨＰＬＣカラム（径１.０×２５cm；Asahipak ＯＤＰ−５０，Asahi chemical）にかけた。カラムを０.１％ＴＦＡで洗浄後、０％から６３％のアセトニトリル濃度勾配を、３４０ml，１７０分間にわたってかけ、ＢＴＣ画分を集めた。この方法により６３０μg の E. coli ＢＴＣが得られた。
E. coli ＢＴＣのＮ末端アミノ酸配列を２０アミノ酸残基まで決定した。ＢＴＣは翻訳開始メチオニンから始まる予想通りのＮ末端を有した分子であることがわかった。
【００３６】
実施例６
参考例４（３）で得られたＮ末端にメチオニンの付加したヒトベータセルリン（Ｍet−ＢＴＣ）２０mgを、５０mＭリン酸緩衝液（pＨ８.０）１６mlに溶解した後に、０.１Ｍ硫酸銅２ml、グリオキシル酸０.９２ｇ、ピリジン２mlの混合液を加え、２５℃で１時間反応した。反応終了後、２Ｍ酢酸−２Ｍ酢酸ナトリウム溶液で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５mmID×６００mmL）に反応液を通液し、平衡化に用いた溶液を６ml／分の流速で展開し、Ｍet−ＢＴＣのジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０mＭ濃度になるように添加して、脱気、窒素ガスシールを行った後、３７℃で２０時間反応した。反応終了後、２０mＭトリス緩衝液（pＨ８.０）で平衡化したセファデックスＧ−２５カラム（２５mmID×６００mmL）に反応液を通液し、平衡化に用いた緩衝液を６ml／分の流速で展開し、Ｎ末端にメチオニンの付加していないヒトベータセルリン（ＢＴＣ）画分をプールした。プールしたＢＴＣ画分をpＨ６.０に調整後、１００mＭリン酸緩衝液（pＨ６.０）で平衡化したＳＰ−セファロース（１０mmID×２００mmL）に吸着した後に、０−１００％Ｂ（Ｂ＝１００mＭリン酸緩衝液＋１Ｍ ＮaＣl、pＨ６.０）の段階勾配で６０分間、２.０ml／分の流速で溶出を行い、ＢＴＣ画分をプールした。さらに、０.１％ＴＦＡで平衡化したＯＤＰ−５０（１０mmID×２５０mmL、昭和電工(株)）に吸着した後、２０−６０％Ｂ（Ｂ＝アセトニトリル／０.１％ＴＦＡ）の段階勾配で４０分間、２ml／分の流速で溶出した。ＢＴＣのフラクションをプールした後、凍結乾燥を行い、ＢＴＣの凍結乾燥粉末を得た。
【００３７】
実施例７：（ＢＴＣの特徴決定）
ａ) ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例６で得られたＢＴＣの１μg を Sample buffer［Laemmli, ネイチャー（Nature），２２７，６８０（１９７０）］に懸濁し、１００ｍＭ ＤＴＴにより１００℃で１分間加熱した後、マルチゲル１５／２５（第一化学薬品(株)）で電気泳動を行った。泳動後のゲルをクーマシーブリリアントブルー（Coomassie brilliant blue）で染色したところ、単一バンドの蛋白が認められ、精製品はほぼ単一であった。結果を〔図３〕に示す。〔図３〕において、Lane １〜２は分子量マーカーおよびＢＴＣを示す。
【００３８】
ｂ) Ｎ末端アミノ酸配列分析
実施例６で得られたＢＴＣを１n mol 用いて、そのＮ末端アミノ酸配列を、気相プロテインシーケンサー（アプライドバイオシステムズ モデル４７７Ａ）により決定した。その結果、ＢＴＣの cＤＮＡの塩基配列から推定したＮ末端アミノ酸配列と一致した。結果を〔表７〕に示す。
【表７】

【００３９】
ｃ) アミノ酸組成分析
実施例６で得られたＢＴＣを約２０μg 用いて、そのアミノ酸組成をアミノ酸分析計（ベックマン システム６３００Ｅ）により決定した。その結果、ＢＴＣの cＤＮＡの塩基配列から推定したアミノ酸組成と一致した。結果を〔表８〕に示す。
【表８】

【００４０】
ｄ) Ｃ末端アミノ酸分析
実施例６で得られたＢＴＣを１５n mol用いて、そのＣ末端アミノ酸をアミノ酸分析計（ベックマン システム６３００Ｅ）により決定した。その結果、得られたＢＴＣの cＤＮＡの塩基配列から推定したＣ末端アミノ酸と一致した。結果を〔表９〕に示す。
【表９】

【００４１】
ｅ) ＢＴＣの生物活性
モレキュラー・セル・バイオロジー、８、５８８（１９８８）に記載の方法により、ＢＡＬＢ／Ｃ３Ｔ３ Ａ３１−７１４クローン４（インターナショナル・ジャーナル・オブ・キャンサー、１２、４６３、（１９７３）を用いた活性測定を行い、実施例６で得られたＢＴＣの精製品が標準品と同等の活性を有することを確認した。
【００４２】
参考例５
メチオニルヒトインターロイキン−２（Ｍet−ＩＬ−２）の製造
特開昭６２−１７１６９９の参考例２に記載の方法に準じてＭet−ＩＬ−２を製造した。
（i）発現用プラスミドの構築
ヒトＩＬ−２遺伝子を有するプラスミド pＩＬＯＴ １３５−８［特開昭６０−１１５５２８の実施例１（vii）参照］を制限酵素Ｈ giＡＩで切断した。得られた１２９４bpＤＮＡ断片をＴ４ポリメラーゼで平滑末端とし、Ｔ４ＤＮＡリガーゼを用いて、Ｅ coＲＩリンカーdＴＧＣＣＡＴＧＡＡＴＴＣＡＴＧＧＣＡ（配列番号：８）を結合させた。得られたＤＮＡをＥ coＲＩで消化し、翻訳開始コドンＡＴＧおよびヒトＩＬ−２遺伝子を有するＤＮＡ断片を得た。
このＤＮＡ断片を、あらかじめＥ coＲＩ−Ｐ stＩ部位を消化した ptrp ７８１［ヌクレイック・アシズ・リサーチ，第１１巻，３０７７頁（１９８３）］にＴ４ＤＮＡリガーゼを用いて挿入した。かくして得られた発現用プラスミド pＴＦ１は trp プロモーターの下流に翻訳開示コドンとヒトＩＬ−２遺伝子を有する（特開昭６２−１７１６９９の〔第４図〕参照）。
プラスミド pＴＦ１を制限酵素Ｓ tuＩで切断し、Ｂ amＨＩリンカーと結合させた。このプラスミドＤＮＡを制限酵素Ｂ amＨＩおよびＥ coＲＩで処理し、ついでＥ coＲＩ−Ｂ amＨＩ部位にλＰＬプロモーターを有するプラスミド pＴＢ２８１に挿入した。かくして得た発現用プラスミドを pＴＢ２８５と命名した（特開昭６２−１７１６９９〔第５図〕参照）。
【００４３】
（ii）形質転換体の製造
上記で得たプラスミド pＴＢ２８５でエシェリヒア コリＮ４８３０をコーエンらの方法［プロシージングス・オブ・ナショナル・アカデミー・オブ・サイエンス（Pro. Natl. Acad. Sci. ＵＳＡ），第６９巻，２１１０頁（１９７２）］に従い形質転換し、上記プラスミドを含有する形質転換体エシェリヒア コリＮ４８３０／pＴＢ２８５を得た。
（iii）形質転換体の培養
形質転換体 E. coli Ｎ４８３０／pＴＢ２８５（ＩＦＯ １４４３７，ＦＥＲＭ ＢＰ−８５２）を２５０ml容フラスコ内のバクト・トリプトン（ディフコ・ラボラトリーズ，アメリカ）１％，バクト・イーストエキス（ディフコ・ラボラトリーズ，アメリカ）０.５％，食塩０.５％およびアンピシリン５０μg／mlを含む液体培地（pＨ７.０）５０mlに接種して３７℃で一晩回転振盪培養した。この培養液をカザミノ酸０.５％，グルコース０.５％およびアンピシリン５０μg／mlを含むＭ９培地２.５L の入った５L 容ジャーファーメンターに移し３５℃で６時間、ついで４２℃でさらに３時間通気撹拌培養して培養液２.５L を得た。この培養液を遠心分離し、菌体を集め、−８０℃で凍結して保存した。
【００４４】
（iv）抽出
凍結菌体２０ｇを７Ｍ塩酸グアニジン０.１Ｍ Tris−ＨＣlを含む抽出液（pＨ７.０）１００mlに均一に懸濁し、４℃で１時間撹拌した後、２８,０００×ｇで２０分間遠心分離し上清を得た。
（v）メチオニルインターロイキン−２蛋白質の部分精製
得られた上清を０.０１Ｍ Tris−ＨＣl 緩衝液（pＨ８.５）に対して透析後、１９,０００×ｇで１０分間遠心分離して得た上清を、０.０１Ｍ Tris−ＨＣl 緩衝液（pＨ８.５）で平衡化したＤＥ５２（ＤＥＡＥ−セルロース，ワットマン社製，イギリス）カラム（５０ml容）に通して蛋白を吸着後、ＮaＣl 濃度直線勾配（０〜０.１５Ｍ ＮaＣl，１L）を作成して、Ｍet−ＩＬ−２を溶出させ、活性画分を得た。
【００４５】
（vi）メチオニルインターロイキン−２蛋白質の精製
上記で得られた活性画分をＹＭ−５メンブラン（アミコン社製，アメリカ）を用いて、５mlに濃縮し、０.１Ｍ Tris−ＨＣl（pＨ８.０）−１Ｍ ＮaＣl 緩衝液で平衡化したセファクリルＳ−２００（ファルマシア製，スウエーデン）カラム（５００ml容）を用いて、ゲルろ過を行った。活性画分４０mlをＹＭ−５メンブランで３mlに濃縮した。得られた濃縮液を、ウルトラポアＲＰＳＣ（アルテックス社製，アメリカ）カラムに吸着させ、トリフルオロ酢酸−アセトニトリル系を溶出溶媒とする高速液体クロマトグラフィーを行った。カラム，ウルトラポアＲＰＳＣ（４.６×７５mm）；カラム温度，３０℃：溶出溶媒Ａ，０.１％トリフルオロ酢酸−９９.９％水；溶出溶媒Ｂ，０.１％トリフルオロ酢酸−９９.９％アセトニトリル；溶出プログラム，０分（６８％＋３２％Ｂ）−２５分（５５％Ａ＋４５％Ｂ）−３５分（４５％Ａ＋５５％Ｂ）−４５分（３０％Ａ＋７０％Ｂ）−４８分（１００％Ｂ）；溶出速度，０.８ml／min；検出波長，２３０nm。
本条件下で保持時間約３９分のＭet−ＩＬ−２画分を集めた。
【００４６】
（vii）ＳＰ−５ＰＷカラムによるＭet−ＩＬ−２の精製
Ｍet−ＩＬ−２を含む０.００５Ｍ酢酸アンモニウム緩衝液（pＨ５.０，蛋白質濃度１.０３mg／ml）０.５mlを、０.０２５Ｍリン酸緩衝液（pＨ７.４）で平衡化した高速液体クロマトグラフィー用ＳＰ−５ＰＷカラム（０.７５×７.５cm；東ソー社製）にのせ、０.０２５Ｍリン酸緩衝液（pＨ７.４）を用いて蛋白質を溶出した。カラム温度は３５℃に、緩衝液の流速は０.５ml／min に設定した。クロマトグラフシステムはバリアン社製５５００型液体クロマトグラフを用いた。
本条件下で保持時間約７０分のＭet−ＩＬ−２画分を集めた。
【００４７】
実施例８
参考例５で得られたメチオニルヒトインターロイキン−２（Ｍet−ＩＬ−２）２０mgを、２０mＭ酢酸アンモニウム緩衝液（pＨ５.０）２７mlに溶解した後、グリオキシル酸１.５５ｇ、２５mＭ硫酸銅３.３７５ml、ピリジン３.３７５mlの混合液を加え、室温で１時間反応した。反応終了後、２０mＭ酢酸アンモニウム緩衝液（pＨ５.０）で平衡化したセファデックスＧ−２５カラム（２５mmID×６００mmL）に３００ml／h の流速で反応液を通液し、Ｍet−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０mＭ濃度になるように添加し、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、２０mＭ酢酸アンモニウム緩衝液（pＨ５.０）で平衡化したセファデックスＧ−２５カラム（２５mmID×６００mmL）に３００ml／h の流速で反応液を通液し、ヒトインターロイキン−２（ＩＬ−２）画分をプールした。プールしたＩＬ−２画分を２５mＭリン酸緩衝液（pＨ７.０）で平衡化したＳＰ−５ＰＷに吸着した後、３０−８０％Ｂ（Ｂ：２５mＭリン酸緩衝液、pＨ８.０）pＨ勾配で２５分間、１.０ml／min の流速で溶出を行い、ＩＬ−２画分をプールした。さらに、▲１▼０.１％ＴＦＡと▲２▼０.１％ＴＦＡ＋８０％アセトニトリル（８０％：２０％）で平衡化したＯＤＰ−５０カラム（４.６mmID×１５０mmL）に吸着した後、２０−１００％Ｂ濃度勾配で１５分間、０.８ml／min の流速で溶出を行い、精製ＩＬ−２を得た。得られたＩＬ−２画分を凍結乾燥し、ＩＬ−２の凍結乾燥粉末を得た。
【００４８】
実施例９（ＩＬ−２の特徴決定）
ａ) ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例８で得られたＩＬ−２の３μg を Sample buffer［Laemmli, ネイチャー（Nature），２２７，６８０（１９７０）］に懸濁した後、１００mＭ ＤＴＴにより１００℃で５分間の還元処理を行い、マルチゲル１０／２０（第一化学薬品）で電気泳動を行った。泳動後のゲルをクマシーブリリアントブルーで染色したところ、標品と同じ移動度を示し、単一バンドであった。結果を〔図４〕に示す。〔図４〕おいて、Lane １〜２はＩＬ−２および分子量マーカーを示す。
【００４９】
ｂ) Ｎ末端アミノ酸配列分析
実施例８で得られたＩＬ−２を１n mol 用いて、そのＮ末端アミノ酸配列を、気相プロテインシーケンサー（アプライドバイオシステム モデル４７７Ａ）により決定した。その結果、ＩＬ−２の cＤＮＡの塩基配列から予想されるＮ末端アミノ酸配列と一致した。結果を〔表１０〕に示す。
【表１０】

【００５０】
ｃ) アミノ酸組成分析
実施例８で得られたＩＬ−２を約２０μg 用いて、そのアミノ酸組成をアミノ酸分析計（ベックマン システム６３００Ｅ）により決定した。その結果、ＩＬ−２の cＤＮＡ塩基配列から予想されるアミノ酸組成と一致した。結果を〔表１１〕に示す。
【表１１】

【００５１】
ｄ) Ｃ末端アミノ酸分析
実施例８で得られたＩＬ−２を１５n mol用いて、そのＣ末端アミノ酸をアミノ酸分析計（ベックマン システム６３００Ｅ）により決定した。その結果、ＩＬ−２の cＤＮＡの塩基配列から予想されるＣ末端アミノ酸と一致した。結果を〔表１２〕に示す。
【表１２】

【００５２】
ｅ) 生物活性
生物活性の測定は、インターロイキン−２依存性細胞を用いる日沼らの方法［バイオケミカル・バイオフィジカル・リサーチ・コミュニケーションズ（Biochem. Biophys. Res. Commun.），１０９，３６３（１９８２）］に従って行い、実施例８で得られたＩＬ−２の精製品が標品と同等の活性を有することを確認した。
【００５３】
参考例６ （Ｔ７プロモーターを用いたヒト成長ホルモン（ｈＧＨ）発現ベクターの構築）
ｈＧＨの構造遺伝子はプラスミドｐＨＧＨ１０７（特公平６−１２９９６に記載、寄託番号ＡＴＣＣ３１５３８およびＡＴＣＣ４００１１）からＥｃｏＲＩおよびＥｃｏＲＶで切断し約０．７５ｋｂの断片を単離した。一方、ｐＥＴ−３Ｃ〔Ｒｏｓｅｎｂｅｒｇ ｅｔ ａｌ．，ジーン（Ｇｅｎｅ）、５６巻、１２５頁（１９８７年）〕をＮｄｅＩおよびＢａｍＨＩで切断し、Ｔ７プロモーターおよびアンピシリン耐性遺伝子を有する約４．６ｋｂの断片を単離した。
両断片をＴ４ＤＮＡポリメラーゼ（ＤＮＡ Ｂｌｕｎｔｉｎｇ ｋｉｔ、宝酒造株式会社製）で平滑末端とし、Ｔ４ＤＮＡリガーゼで連結したのち、大腸菌ＪＭ１０９に導入し、アンピシリン耐性を指標として形質転換体を選択した。出現したコロニーの中から１２個を拾い、これらからプラスミドを調製し制限酵素ＰｓｔＩによる切断パターンを調べたところ、６個のコロニーからのプラスミドにおいてｈＧＨ遺伝子が正しい方向で挿入されていることがわかった。この中の１株から得られたプラスミドをｐＴＧＡ２０１と命名した。
【００５４】
参考例７ （大腸菌でのＭｅｔ−ｈＧＨの発現）
大腸菌ＪＭ１０９を、Ｔ７ファージのＲＮＡポリメラーゼ遺伝子で組み換えられているラムダファージ（スチュディエ、スプラ）で溶原化した。その後、参考例６で得られたｈＧＨ発現ベクターｐＴＧＡ２０１をこの大腸菌ＪＭ１０９（ＤＥ３）へ導入し、大腸菌ＪＭ１０９（ＤＥ３）／ｐＴＧＡ２０１を得た。
この形質転換細胞を、５０μｇ／ｍｌのアンピシリンを含むＬＢ培地（１％ペプトン、０．５％酵母エキス、０．５％塩化ナトリウム）１リットルを含む２リットル容フラスコ中で３０℃、１６時間振とう培養した。得られた培養液を、０．０２％ニューポールＬＢ−６２５（消泡剤；三洋化成工業製）および５０μｇ／ｍｌのアンピシリンを含む２０リットルのＬＢ培地を仕込んだ５０リットル容発酵槽へ移植して、３７℃、６時間通気撹拌培養した。この培養液を３６０リットルの主発酵培地（１．６８％リン酸一水素ナトリウム、０．３％リン酸二水素カリウム、０．１％塩化アンモニウム、０．０５％塩化ナトリウム、０．０２４６％硫酸マグネシウム、０．０２％ニューポールＬＢ−６２５、０．０００５％塩酸チアミン、１．５％ブドウ糖、１．５％カザミノ酸）を仕込んだ５００リットル容発酵槽に移植して、３７℃で通気撹拌培養を開始した。培養液の濁度が約５００クレット単位になった時点で、５．９５ｍｇ／リットル分のイソプロピル−β−Ｄ−チオガラクトピラノシド（ＩＰＴＧ）を添加し、さらに培養を続け、４時間後に培養液を遠心分離して、約４．５ｋｇの湿菌体を得、−８０℃に凍結保存した。
上記の形質転換大腸菌ＪＭ１０９（ＤＥ３）／ｐＴＧＡ２０１は、受託番号ＦＥＲＭ ＢＰ−５６３２として通商産業省工業技術院生命工学工業技術研究所（ＮＩＢＨ）に寄託され、また受託番号ＩＦＯ １６００１として財団法人発酵研究所（ＩＦＯ）に寄託されている。
【００５５】
参考例８ （Ｍｅｔ−ｈＧＨの活性化）
参考例７で得られた菌体２ｋｇに５０ｍＭトリス／ＨＣｌ、８Ｍグアニジン塩酸塩溶液（ｐＨ８．０）６リットルを加えて菌体を溶解後、遠心分離（１００００ｒｐｍ、１２０分間）を行った。得られた上澄液６リットルに５０ｍＭトリス／ＨＣｌ、０．２８ｍＭＧＳＳＧ、１．４ｍＭＧＳＨ、０．７Ｍアルギニン（ｐＨ８．０）１８リットルを加えてｐＨ８．０に調整した後、４℃で５日間活性化を行った。
【００５６】
参考例９ （Ｍｅｔ−ｈＧＨの精製）
参考例８で活性化の終了した再生液をペリコンカセットシステム（ＰＴＧＣ膜、ミリポア社）で、２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）を加えながら電気伝導度が１０ｍＳ以下になるまで脱塩、濃縮を行った後、得られた濃縮液を遠心分離（１００００ｒｐｍ、６０分間）し、濃縮液の上清５リットルを得た。ついでこの液を２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）で平衡化したＤＥＡＥ−トヨパール６５０Ｍカラム（２０ｃｍφ×８４ｃｍ、東ソー社）に吸着させ、十分に洗浄した後、０〜２５％Ｂ（Ｂ＝２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素、１Ｍ塩化ナトリウム、ｐＨ８．０）の濃度勾配で１００分間、３００ｍｌ／分の流速で溶出を行い、Ｍｅｔ−ｈＧＨ画分として１０リットルの溶出液を得た。さらに、この溶出液をペリコンカセットシステム（ＰＴＧＣ膜、ミリポア社）で濃縮、脱塩した。更に、高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２１ｃｍ×３０ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、３２０ｍｌ／分の流速で溶出を行い、Ｍｅｔ−ｈＧＨ画分６リットルを得た。この溶出液に２Ｍトリス／ＨＣｌ（ｐＨ７．８）溶液３００ｍｌ加えてｐＨ７．２に調整し、ついでペリコンカセットシステム（ＰＴＧＣ膜、ミリポア社）で濃縮、脱塩し、９，９７９ミリグラムのＭｅｔ−ｈＧＨを得た。
【００５７】
実施例１０（Ｎ末端Ｍｅｔの除去）
参考例９で得たＭｅｔ−ｈＧＨ溶液１６５０ｍｌに２．５Ｍグリオキシル酸、３５ｍＭ硫酸銅、６Ｍピリジン溶液４１３ｍｌを加えよく撹拌した後２５℃で６０分間反応させた。次いで、２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１１．３ｃｍφ×１２５ｃｍ、ファルマシア社）に３リットル／ｈの流速で通液し、平衡化と同じ緩衝液を用いて展開し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を、よく撹拌しながら直接４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭｏ−フェニレンジアミン、３Ｍ尿素溶液４リットル中に加えた。溶出終了後、この反応溶液８リットルを４℃に３日間静置した。静置後、ペリコンカセットシステム（ＰＴＧＣ膜、ミリポア社）で４リットルに濃縮し、２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１１．３ｃｍφ×１４０ｃｍ、ファルマシア社）に３リットル／ｈの流速で通液し、ｈＧＨ画分４．７リットルを集めた。更に、高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２１ｃｍ×３０ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、３２０ｍｌ／分の流速で溶出を行い、ｈＧＨ画分１０リットルを得た。このｈＧＨ画分に２Ｍトリス／ＨＣｌ（ｐＨ７．８）溶液を５００ｍｌ加えてｐＨ７．２に調整後、ミニタンII（ＰＴＧＣ膜、ミリポア社）で濃縮し、この濃縮液５００ｍｌを蒸留水で平衡化したセファクリルＳ−１００カラム（１１３．ｃｍφ×５０ｃｍ、ファルマシア社）に２リットル／ｈの流速で通液、展開しｈＧＨ画分１６５１ｍｌを得た。更に、この溶液をミリパック６０（ミリポア社）でろ過し、ｈＧＨ溶液１４８７ｍｌ（３３０９ｍｇのｈＧＨ）を得た。
【００５８】
実施例１１ （ｈＧＨの特徴決定）
（ａ）ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例１０で得られたｈＧＨに１００ｍＭＤＴＴを含むサンプルバッファー［Laemmli, Nature, 227, 680（1970）］を等量加えてよく撹拌し、９５℃で２分間加熱後、マルチゲル１０／２０（第一化学薬品）で電気泳動を行った。泳動後のゲルをクーマシー・ブリリアント・ブルー（Coomassie brilliant blue）で染色した結果、約２２ｋｄに単一バンドが認められたことから、精製ｈＧＨはほぼ単一であることが確認された（図５）。図５において、レーン１〜２は分子量マーカーおよびｈＧＨを示す。
【００５９】
（ｂ）アミノ酸組成分析
アミノ酸組成をアミノ酸分析計（Ｌ−８５００Ａ，日立）を用いて決定した。その結果、得られたｈＧＨのアミノ酸組成はｃＤＮＡの塩基配列から推定されるアミノ酸組成と一致した（表１３）。
【表１３】

【００６０】
（ｃ）Ｎ末端アミノ酸配列分析
Ｎ末端アミノ酸配列を気相プロテインシーケンサー（アプライドバイオシステムズ社、モデル４７７Ａ）を用いて決定した。その結果、得られたｈＧＨのＮ末端アミノ酸配列はｃＤＮＡの塩基配列から推定されたｈＧＨのＮ末端アミノ酸配列と一致した（表１４）。
【表１４】

【００６１】
（ｄ）Ｃ末端アミノ酸分析
Ｃ末端アミノ酸をアミノ酸分析計（Ｌ−８５００Ａ，日立）を用いて決定した。得られたｈＧＨのＣ末端アミノ酸はｃＤＮＡの塩基配列から推定されたＣ末端アミノ酸と一致した（表１５）。
【表１５】

【００６２】
実施例１２ （ｈＧＨの活性測定）
実施例１０で得られた精製ｈＧＨのＮｂ２細胞〔ジャーナル・オブ・クリニカル・エンドクリノロジー・アンド・メタボリズム、５１巻、１０５８頁（１９８０）〕に対する増殖促進効果は、標準品（ケミコンインターナショナル社、Temecula，California，USA）と同等であった。
【００６３】
実施例１３ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ塩化ニッケル、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭｏ−フェニレンジアミン溶液を加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００６４】
実施例１４ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ塩化コバルト、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭｏ−フェニレンジアミン溶液を加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００６５】
実施例１５ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸亜鉛、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭｏ−フェニレンジアミン溶液を加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００６６】
実施例１６ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ酢酸銅、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭｏ−フェニレンジアミン溶液を加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００６７】
実施例１７ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭトリレン−３，４−ジアミン溶液を加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００６８】
実施例１８ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭ４−クロロ−ｏフェニレンジアミン溶液を加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００６９】
実施例１９ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、８０ｍＭ３，４−ジアミノ安息香酸溶液を加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００７０】
実施例２０ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、８Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が１０ｍｇ／ｍｌになるように濃縮した。この溶液１ｍｌに４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、６Ｍ尿素（ｐＨ７．０）溶液１ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、４Ｍ尿素（ｐＨ８．５）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に４０ｍＭになるようにシステアミンを加えよく撹拌した後３７℃で１５時間反応した。反応後、２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００７１】
実施例２１ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、３Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が５ｍｇ／ｍｌになるように濃縮した。この溶液２ｍｌに４Ｍ酢酸ナトリウム、０．８Ｍ酢酸、０．４Ｍグリオキシル酸、１０ｍＭ硫酸銅、２．５Ｍ尿素溶液２ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、３Ｍ尿素、８０ｍＭｏ−フェニレンジアミン溶液を加えよく撹拌した後４℃で３日間反応した。反応後、２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００７２】
実施例２２ （Ｎ末端Ｍｅｔの除去）
参考例９と同じ方法で得たＭｅｔ−ｈＧＨ溶液を限外濾過システム（ダイアフローメンブレンＹＭ１０、４３ｍｍ、アミコン社）を使って２０ｍＭトリス／ＨＣｌ、３Ｍ尿素（ｐＨ８．０）に置換するとともにＭｅｔ−ｈＧＨの濃度が５ｍｇ／ｍｌになるように濃縮した。この溶液２ｍｌに１Ｍイミダゾール、０．５Ｍグリオキシル酸、２０ｍＭ硫酸銅、２．５Ｍ尿素溶液２ｍｌを加えよく撹拌した後室温で６０分間反応させた。次いで、この反応液を２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×３０ｃｍＬ、ファルマシア社）に通液し、溶出してきたＭｅｔ−ｈＧＨのジケトン体画分を集め、この溶出液に等量の４Ｍ酢酸、４Ｍ酢酸ナトリウム、３Ｍ尿素、８０ｍＭｏ−フェニレンジアミン溶液を加えよく撹拌した後４℃で３日間反応した。反応後、２０ｍＭトリス／ＨＣｌ、２．５Ｍ尿素（ｐＨ８．０）で平衡化したセファデックスＧ−２５カラム（１０ｍｍＩＤ×４０ｃｍＬ、ファルマシア社）に通液し、ｈＧＨ画分を集めた後、更に高速液体クロマトグラフ法（ギルソンＨＰＬＣシステム、ギルソン社）により、この溶液をＤＥＡＥ−５ＰＷカラム（２．１５ｃｍ×１５ｃｍ、東ソー社）に通液吸着させた後、Ａ＝５０ｍＭトリス／ＨＣｌ＋２．５Ｍ尿素（ｐＨ８．０）、Ｂ＝５０ｍＭＭＥＳ［２−（Ｎ−モルホリノ）エタンスルホン酸］＋２．５Ｍ尿素（ｐＨ４．０）とによる７０〜８５％ＢのｐＨ勾配で、７０分間、７．５ｍｌ／分の流速で溶出を行い、ｈＧＨを得た。
【００７３】
実施例２３ （Ｎ末端Ｍｅｔの除去）
参考例４で得られたＮ末端にＭｅｔの付加したＭｅｔ−ＢＴＣ１０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０．２５ｇ、ピリジン０．５ｍｌの混合液（ｐＨ５．０）を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Ｓｅｐｈａｄｅｘ）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度となるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、反応液を５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた緩衝液を６ｍｌ／分の流速で展開し、Ｎ末端にＭｅｔの付加していないＢＴＣ画分をプールした。プールしたＢＴＣ画分をｐＨ６．０に調整後、１００ｍＭリン酸緩衝液＋２００ｍＭ ＮａＣｌ（ｐＨ５．０）で平衡化したＳＰ−５ＰＷ（７．５ｍｍＩＤ×７５ｍｍＬ）、東ソー（株））に吸着した後、０〜１００％Ｂ（Ｂ＝１００ｍＭリン酸緩衝液＋２００ｍＭ ＮａＣｌ、ｐＨ９．０）の段階勾配で３０分間、０．８ｍｌ／分の流速で溶出を行い、ＢＴＣ画分をプールした、さらに、ＢＴＣ画分を０．１％ＴＦＡで平衡化したＯＤＰ−５０（１０ｍｍＩＤ×２５０ｍｍＬ）、昭和電工（株））に吸着した後、２０〜６０％Ｂ（Ｂ＝８０％アセトニトリル／０．１％ＴＦＡ）の段階勾配で４０分間、２ｍｌ／分の流速で溶出した。ＢＴＣのフラクションをプールした後、凍結乾燥を行い、ＢＴＣ約７６０μｇを得た。
【００７４】
実施例２４ （ＢＴＣの特徴決定）
（ａ）ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例２３で得られたＢＴＣを１００ｍＭ ＤＴＴを添加したＳａｍｐｌｅ ｂｕｆｆｅｒ［Laemmli, Nature, 227, 680 (1970)］に懸濁し、９５℃で１分間加熱した後、マルチゲル１５／２５（第一化学薬品）で電気泳動を行った。泳動後のゲルをクーマシー・ブリリアント・ブルー（Coomassie brilliant blue）で染色した結果、約１６Ｋｄに単一バンドの蛋白が認められた。このことから、精製ＢＴＣはほぼ単一であり、ＤＴＴによる還元条件下でも強固な二量体を形成していることが判った（図６）。図６において、レーン１〜２は分子量マーカーおよびＢＴＣを示す。
【００７５】
（ｂ）アミノ酸組成分析
アミノ酸組成をアミノ酸分析計（ベックマンシステム６３００Ｅ）を用いて決定した。その結果、ＢＴＣのｃＤＮＡの塩基配列から推定されたアミノ酸組成と一致した（表１６）。
【表１６】

【００７６】
（ｃ）Ｎ末端アミノ酸配列分析
Ｎ末端アミノ酸配列を気相プロテインシーケンサー（アプライドバイオシステムモデル４７７Ａ）を用いて決定した。その結果、ＢＴＣのｃＤＮＡの塩基配列から推定されたＢＴＣのＮ末端アミノ酸配列と一致した（表１７）。
【表１７】

【００７７】
（ｄ）Ｃ末端アミノ酸分析
Ｃ末端アミノ酸をアミノ酸分析計（ベックマンシステム６３００Ｅ）を用いて決定した。得られたＢＴＣのＣ末端アミノ酸はｃＤＮＡの塩基配列から推定したＣ末端アミノ酸と一致した（表１８）。
【表１８】

【００７８】
実施例２５ （ＢＴＣの生物活性）
モレキュラー・セル・バイオロジー、８巻、５８８頁（１９８８年）に記載の方法により、ＢＡＬＢ／ｃ ３Ｔ３ Ａ３１−７１４クローン４（インターナショナル・ジャーナル・オブ・キャンサー、１２巻、４６３頁（１９７３年））を用いた活性測定を行い、実施例２３で得られた精製ＢＴＣが標準品（特開平６−８７８９４の実施例１３に記載の精製ＢＴＣＩ）と同等の細胞増殖促進活性を有することを確認した。
【００７９】
実施例２６
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、トリレン−３，４−ジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８０】
実施例２７
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、２，３−ジアミノフェノールを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８１】
実施例２８
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、３，４−ジアミノ安息香酸を４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８２】
実施例２９
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、４−クロロ−ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８３】
実施例３０
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分にシステアミン４０ｍＭ濃度になるように添加し、ｐＨ８．５に調製した後、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８４】
実施例３１
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸ニッケル、６Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８５】
実施例３２
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ塩化コバルト、６Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８６】
実施例３３
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸亜鉛、６Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８７】
実施例３４
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ酢酸銅０．５ｍｌ、グリオキシル酸０．２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８８】
実施例３５
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、０．８Ｍ酢酸、０．４Ｍグリオキシル酸、１０ｍＭ硫酸銅、３Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００８９】
実施例３６
参考例４で得られたＮ末端にメチオニンの付加したＭｅｔ−ＢＴＣ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、１Ｍイミダゾール、０．５Ｍグリオキシル酸、２０ｍＭ硫酸銅、３Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＢＴＣのジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例２３と同様の方法で精製を行い、ＢＴＣを得た。
【００９０】
実施例３７
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０．２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、反応液を５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた緩衝液を６ｍｌ／分の流速で展開し、Ｎ末端にメチオニンの付加していないＮＴ−３画分をプールした。プールしたＮＴ−３画分をｐＨ６．０に調整後、１００ｍＭリン酸緩衝液＋２００ｍＭ ＮａＣｌ（ｐＨ５．０）で平衡化したＳＰ−５ＰＷ（２１．５ｍｍＩＤ×１５０ｍｍＬ、東ソー（株））に吸着した後、０−１００％Ｂ（Ｂ＝１００ｍＭリン酸緩衝液＋２００ｍＭ ＮａＣｌ、ｐＨ９．０）の段階勾配で５５分間、６ｍｌ／分の流速で溶出を行い、ＮＴ−３画分をプールした。さらに、ＮＴ−３画分を０．１％ＴＦＡで平衡化したＯＤＰ−５０（１０ｍｍＩＤ×２５０ｍｍＬ、昭和電工（株））に吸着した後、２０−６０％Ｂ（Ｂ＝８０％アセトニトリル／０．１％ＴＦＡ）の段階勾配で４０分間、２ｍｌ／分の流速で溶出した。ＮＴ−３のフラクションをプールした後、凍結乾燥を行い、ＮＴ−３約６００μｇ得た。
【００９１】
実施例３８（ＮＴ−３の特徴決定）
ａ）ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例３７で得られたＮＴ−３を１００ｍＭ ＤＴＴを含む Sample buffer［Laemmli, Nature, 227, 680(1970)］に懸濁し１００℃で１分間加熱した後、マルチゲル１５／２５（第一化学薬品）で電気泳動を行った。泳動後のゲルをクーマシー ブリリアント ブルー（Coomassie brilliant blue）で染色したところ、単一バンドの蛋白が認められ、精製品はほぼ単一であった（図７）。図７において、レーン１〜２は分子量マーカーおよびＮＴ−３を示す。
【００９２】
Ｎ末端アミノ酸配列分析
実施例３７で得られたＮＴ−３のＮ末端アミノ酸配列を気相プロテインシーケンサー（アプライドバイオシステムズ モデル４７７Ａ）を用いて決定した。その結果、得られたＮＴ−３のＮ末端アミノ酸配列はｃＤＮＡの塩基配列から推定したＮＴ−３のＮ末端アミノ酸配列と一致した。（表１９）
【表１９】

【００９３】
ｂ）アミノ酸組成分析
実施例３７で得られたＮＴ−３のアミノ酸組成をアミノ酸分析計（ベックマンシステム６３００Ｅ）を用いて決定した。その結果、ＮＴ−３のｃＤＮＡの塩基配列から推定したアミノ酸組成と一致した（表２０）
【表２０】

【００９４】
Ｃ末端アミノ酸分析
実施例３７で得られたＮＴ−３のＣ末端アミノ酸をアミノ酸分析計（ベックマン システム６３００Ｅ）を用いて決定した。得られたＮＴ−３のＣ末端アミノ酸配列はｃＤＮＡの塩基配列から推定したＣ末端アミノ酸と一致した（表２１）。
【表２１】

【００９５】
実施例３９（ＮＴ−３の生物活性の測定）
実施例３７で得られたＮＴ−３について、ＤＲＧを用いた生物活性測定を行い、ＣＨＯ細胞より得られたＮＴ−３と同等の活性を有することを確認した。
【００９６】
実施例４０
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、トリレン−３，４−ジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【００９７】
実施例４１
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、２，３−ジアミノフェノールを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【００９８】
実施例４２
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、３，４−ジアミノ安息香酸を４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【００９９】
実施例４３
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、４−クロロ−ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１００】
実施例４４
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ硫酸銅０．５ｍｌ、グリオキシル酸０.２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分にシステアミンを４０ｍＭ濃度になるように添加し、ｐＨ８．５に調製した後、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１０１】
実施例４５
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸ニッケル、６Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１０２】
実施例４６
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ塩化コバルト、６Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１０３】
実施例４７
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸亜鉛、６Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１０４】
実施例４８
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ４０ｍｇを３Ｍ尿素溶液４ｍｌに溶解した後、５０ｍＭ酢酸銅０．５ｍｌ、グリオキシル酸０．２５ｇ、ピリジン０．５ｍｌの混合液を加え、２５℃で１時間反応した。反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１０５】
実施例４９
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、４Ｍ酢酸ソーダ、０．８Ｍ酢酸、０．４Ｍグリオキシル酸、１０ｍＭ硫酸銅、３Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１０６】
実施例５０
参考例３で得られたＮ末端にメチオニンの付加したＭｅｔ−ＮＴ−３ ２０ｍｇを３Ｍ尿素溶液２ｍｌに溶解した後、１Ｍイミダゾール、０．５Ｍグリオキシル酸、２０ｍＭ硫酸銅、３Ｍ尿素の溶液２ｍｌを添加し、２５℃で１時間反応した。
反応終了後、反応液を２．５Ｍ尿素＋５０ｍＭリン酸緩衝液（ｐＨ６．０）で平衡化したセファデックス（Sephadex）Ｇ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に通液し、平衡化に用いた溶液を６ｍｌ／分の流速で展開し、Ｍｅｔ−ＮＴ−３のジケトン体画分をプールした。続いてこの画分に等量の４Ｍ酢酸−４Ｍ酢酸ナトリウム溶液を加えた後、ｏ−フェニレンジアミンを４０ｍＭ濃度になるように添加して、脱気、窒素ガスシールを行い、３７℃で１５時間反応した。反応終了後、実施例３７と同様の方法で精製を行い、ＮＴ−３を得た。
【０１０７】
変性Ｍｅｔ−ＮＴ−３の活性化および活性化されたＭｅｔ−ＮＴ−３の精製は例えば以下の方法により行うこともできる。すなわち、参考例３（５）で得られたペレット溶液を遠心分離（１００００ｒｐｍ）した後、上澄液を１．８Ｍ尿素、０．２Ｍ Ａｒｇ、０．２ｍＭ ＧＳＳＧ、１．０ｍＭ ＧＳＨ、１００ｍＭリン酸緩衝液（ｐＨ８．５）で約２０倍に希釈し、４℃、４週間で変性Ｍｅｔ−ＮＴ−３のリフォールディングを行うことができる。また、リフォールディング終了後の再生液をｐＨ６．０に調整し、１００ｍＭ リン酸緩衝液(ｐＨ６．０)で平衡化したＳＰ−セファロースカラム（２２ｍｍＩＤ×１２０ｍｍＬ）に吸着した後、４００ｍＭ ＮａＣｌ／１００ｍＭ リン酸緩衝液（ｐＨ６．０）で溶出し、Ｍｅｔ−ＮＴ−３を含むフラクションをプールし、続いて０．１％ＴＦＡで平衡化したＯＤＰ−５０（２１．５ｍｍＩＤ×３００ｍｍＬ）（昭和電工（株））に吸着し、０〜８０％Ｂ（Ｂ＝アセトニトリル／０．１％ＴＦＡ）の段階勾配で６０分間、５ｍｌ／分の流速で溶出して得られるＮＴ−３のフラクションを凍結乾燥して、Ｍｅｔ−ＮＴ−３の粉末を得ることができる。さらに、上述のアルギニンの代わりに、シトルリン、アラニン、バリン、リジン、アスパラギン酸などを用いてリフォールディングを行うこともできる。
【０１０８】
実施例５１
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ塩化ニッケル３．３７５ｍｌ、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、６Ｍ尿素の溶液、３．３７５ｍｌを加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１０９】
実施例５２
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ塩化コバルト３．３７５ｍｌ、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、６Ｍ尿素の溶液、３．３７５ｍｌを加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１０】
実施例５３
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ硫酸亜鉛３．３７５ｍｌ、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、６Ｍ尿素の溶液、３．３７５ｍｌを加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１１】
実施例５４
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ酢酸銅３．３７５ｍｌ、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、６Ｍ尿素の溶液、３．３７５ｍｌを加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１２】
実施例５５
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ硫酸銅３．３７５ｍｌ、ピリジン３．３７５ｍｌの混合液を加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分をｐＨ８．５に調製した後、システアミンを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１３】
実施例５６
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ硫酸銅３．３７５ｍｌ、ピリジン３．３７５ｍｌの混合液を加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にトリレン−３，４−ジアミンを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１４】
実施例５７
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ硫酸銅３．３７５ｍｌ、ピリジン３．３７５ｍｌの混合液を加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分に４−クロロ−ｏ−フェニレンジアミンを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１５】
実施例５８
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ硫酸銅３．３７５ｍｌ、ピリジン３．３７５ｍｌの混合液を加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分に３，４−ジアミノ安息香酸を４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１６】
実施例５９
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、グリオキシル酸１．５５ｇ，０．１Ｍ硫酸銅３．３７５ｍｌ、ピリジン３．３７５ｍｌの混合液を加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分に２，３−ジアミノフェノールを４０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１７】
実施例６０
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、０．５Ｍグリオキシル酸、２０ｍＭ硫酸亜鉛、４０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）、６Ｍ尿素の溶液を等量加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１８】
実施例６１
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、０．５Ｍグリオキシル酸、２０ｍＭ酢酸銅、４０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）、６Ｍ尿素の溶液を等量加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１１９】
実施例６２
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、０．５Ｍグリオキシル酸、２０ｍＭ塩化ニッケル、４０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）、６Ｍ尿素の溶液を等量加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１２０】
実施例６３
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、０．５Ｍグリオキシル酸、２０ｍＭ塩化コバルト、４０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）、６Ｍ尿素の溶液を等量加え、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１２１】
実施例６４
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、３Ｍ尿素を含む２０ｍＭトリスＨＣｌ（ｐＨ８）、２Ｌに対して透析した。この透析内液に１Ｍイミダゾール、０．５Ｍグリオキシル酸、２０ｍＭ硫酸銅、２．５Ｍ尿素溶液を等量混ぜ、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１２２】
実施例６５
参考例５で得られたＮ末端にメチオニンの付加したヒトインターロイキン２（Ｍｅｔ−ＩＬ−２）２０ｍｇを２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）２７ｍｌに溶解した後、３Ｍ尿素を含む２０ｍＭトリスＨＣｌ（ｐＨ８）、２Ｌに対して透析した。この透析内液に４Ｍ酢酸ナトリウム、０．４Ｍグリオキシル酸、１０ｍＭ硫酸銅、０．８Ｍ酢酸、２．５Ｍ尿素溶液を等量混ぜ、室温で１時間反応した。反応終了後、反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５．０）で平衡化したセファデックスＧ−２５カラム（２５ｍｍＩＤ×６００ｍｍＬ）に３００ｍｌ／ｈの流速で通液し、Ｍｅｔ−ＩＬ−２のジケトン体画分をプールした。続いてこの画分にｏ−フェニレンジアミンを２０ｍＭ濃度になるよう添加して、脱気、窒素ガスシールを行った後、３７℃で２１時間反応した。反応終了後、実施例８の方法に従い、ＩＬ−２の凍結乾燥粉末を得た。
【０１２３】
参考例１０
大腸菌ＭＭ２９４（ＤＥ３）／ｐＥ−Ｃ３５ＰＴＨ（ＩＦＯ１５２１３；ＦＥＲＭ ＢＰ−３５０８）（特開平５−３０４９７６号）を１％バクトトリプトン、０．５％イーストエキス、０．５％塩化ナトリウム、５０μｇ／ｍｌのアンピシリンを含む液体培地（ｐＨ７）１Ｌに接種し、３０℃で回転振とう培養した。この培養液１Ｌを１％バクトトリプトン、０．５％イーストエキス、０．５％塩化ナトリウムを含む液体培地（ｐＨ７）１９Ｌに接種し、３０℃で８時間、通気撹拌培養した。この培養液をＭ−９培地（１．５％カザミノ酸、１．５％グルコース、０．０００５％ビタミンＢ₁）３４３Ｌの入った５００Ｌ容発酵槽へ１７Ｌ移し、３７℃で７時間の通気撹拌培養を行い、約３５０Ｌの培養液を得た。この培養液を遠心分離して、約４ｋｇの湿菌体を取得し、−８０℃に凍結保存した。
上記の大腸菌ＭＭ２９４（ＤＥ３）／ｐＥ−Ｃ３５ＰＴＨは、受託番号ＩＦＯ
１５２１３として財団法人発酵研究所（ＩＦＯ）に寄託されている。
【０１２４】
参考例１１
凍結菌体１０ｇに７Ｍ塩酸グアニジンを含む２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）４０ｍｌを加え、菌体を溶解した後、遠心分離（１０，０００ｒｐｍ、６０分）を行い、約４０ｍｌの菌体抽出液を得た。この菌体抽出液を約２Ｌの２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）で希釈した後、遠心分離（４，２００ｒｐｍ、２０分）を行い、約２Ｌの遠心上澄液を得た。この上澄液をＳＰ−トヨパール６５０Ｍカラム（５ｃｍ×３０ｃｍ）に通液し、吸着、洗浄した後、１Ｍ塩化ナトリウムを含む２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）で溶出した。この溶出液をＯＤＳ−１２０Ｔカラム（２１．５ｃｍ×３０ｃｍ、東ソー）及び▲１▼０．１％トリフルオロ酢酸と▲２▼０．１％トリフルオロ酢酸を含む８０％アセトニトリルとによる濃度勾配溶出法を用いた高速液体クロマトグラフィーにより吸着、溶出し、約３０ｍｌのＭｅｔ〔Ｃｙｓ³⁵〕ＰＴＨ（１−８４）画分を得た。この液を５Ｌの６Ｍ尿素を含む０．１Ｍ酢酸に対して透析し、透析液を得た。得られた透析液に約４ｍｇのＤＭＡＰ−ＣＮ（１−シアノ−４−ジメチルアミノピリジニウムテトラフルオロボレート）を加え、室温で１５分間反応し、Ｍｅｔ−〔Ｃｙｓ(ＣＮ)³⁵〕ＰＴＨ（１−８４）を得た。得られた反応液をＳＰ−トヨパール６５０Ｍ（２．０ｃｍ×３０ｃｍ）に通液し、吸着した後、１００ｍＭ塩化ナトリウムを含む、２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）で洗浄を行い、試薬を除去した後、１Ｍ塩化ナトリウムを含む、２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）で溶出した。この溶出液を５Ｌの６Ｍ尿素に対して透析し、約８ｍｌの透析液を得た。得られた透析液を氷冷し、４００μｌの１Ｎ水酸化ナトリウムを加え、０℃で１０分間反応した。得られた反応液を２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）で平衡化したトヨパールＨＷ−５０Ｆ（２ｃｍ×５０ｃｍ）を用いてゲルろ過を行い、ＭｅｔＰＴＨ（１−３４）画分をプールした。得られたＭｅｔＰＴＨ（１−３４）画分をＯＤＳ−１２０Ｔカラム（２１．５ｃｍ×３０ｃｍ、東ソー）及び▲１▼０．１％トリフルオロ酢酸と▲２▼０．１％トリフルオロ酢酸を含む８０％アセトニトリルとによる濃度勾配溶出法を用いた高速液体クロマトグラフィーにより吸着、溶出し、約５０ｍｌのＭｅｔＰＴＨ（１−３４）画分を得た。この溶出液を凍結乾燥し、約５ｍｇのＭｅｔＰＴＨ（１−３４）を得た。
【０１２５】
実施例６６
参考例１１で得られたＮ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを蒸留水１ｍｌに溶解した後、グリオキシル酸４６．４ｍｇ、硫酸銅２．５ｍｇ、ピリジン９４．８ｍｇの混合液を加え、室温で１時間反応した。反応終了後、遠心分離を行い、その上清をＯＤＰ−５０カラム（１ｃｍ×２５ｃｍ、昭和電工）及び▲１▼１０％アセトニトリルを含む２０ｍＭ酢酸アンモニウム（ｐＨ５）と▲２▼６０％アセトニトリルを含む２０ｍＭ酢酸アンモニウム（ｐＨ５）とによる濃度勾配溶出法を用いた高速液体クロマトグラフィーにより吸着、溶出し約５．４ｍｌのＭｅｔ−ＰＴＨ（１−３４）のジケトン体画分をプールした。次にこの画分を凍結乾燥し、得られた凍結乾燥品に蒸留水を加え、４ｍｌとした後、８０ｍＭのｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの混合液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、反応液をＯＤＰ−５０カラム（１ｃｍ×２５ｃｍ、昭和電工）及び▲１▼０．１％トリフルオロ酢酸と▲２▼０．１％トリフルオロ酢酸を含む８０％アセトニトリルとによる濃度勾配溶出法を用いた高速液体クロマトグラフィーにより吸着、溶出を行い、ＰＴＨ（１−３４）画分をプールした。プールしたＰＴＨ（１−３４）画分を２０ｍＭ酢酸アンモニウム（ｐＨ５）で５倍希釈し、２Ｍ尿素を含む２０ｍＭ酢酸アンモニウム緩衝液（ｐＨ５）で平衡化したＳＰ−５ＰＷカラム（７．５ｍｍ×７５ｍｍ、東ソー）に吸着した後、０−５０％Ｂ（Ｂ：２０ｍＭＭＥＳ−２Ｍ尿素−０．５Ｍ塩化ナトリウム、ｐＨ８）濃度勾配で３０分間、０．８ｍｌ／分の流速で行い、ＰＴＨ（１−３４）画分をプールした。この画分を１Ｌ蒸留水に対して透析し、凍結乾燥を行い、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１２６】
実施例６７（ＰＴＨ（１−３４）の特徴決定）
ａ）ＳＤＳポリアクリルアミドゲル電気泳動を用いた分析
実施例６６で得られたＰＴＨ（１−３４）をサンプルバッファ［NOVEX JAPAN］に懸濁した後、Peptide-PAGE mini［TEFCO］で電気泳動を行った。泳動後のゲルをクーマシー ブリリアント ブルーで染色したところ、単一バンドであった（図８）。図８において、レーン１〜２は分子量マーカーおよびＰＴＨ（１−３４）を示す。
【０１２７】
ｂ）Ｎ末端アミノ酸配列分析
Ｎ末端アミノ酸配列を気相プロテインシーケンサー（アプライドバイオシステム モデル４７７Ａ）を用いて決定した。その結果、ＰＴＨ（１−３４）の塩基配列から予想されるＮ末端アミノ酸配列と一致した。（表２２）
【表２２】

【０１２８】
ｃ）アミノ酸組成分析
アミノ酸組成をアミノ酸分析計（日立Ｌ−８５００Ａ Amino Acid Analyzer）を用いて決定した。その結果、ＰＴＨ（１−３４）のｃＤＮＡ塩基配列から予想されるアミノ酸組成と一致した（表２３）
【表２３】

【０１２９】
ｄ）Ｃ末端アミノ酸分析
Ｃ末端アミノ酸をアミノ酸分析計（日立Ｌ−８５００Ａ Amino Acid Analyzer）を用いて決定した。その結果、ＰＴＨ（１−３４）の塩基配列から予想されるＣ末端アミノ酸と一致した。（表２４）
【表２４】

生物活性の測定は骨芽細胞様細胞株ＭＣ３Ｔ３−Ｅ１細胞を用いる方法〔Nakagawa ，S ら、BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONDS，200，1735−1741（1994）〕に従って行い、標品と同等の活性を有することを確認した。
【０１３０】
実施例６８
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ塩化ニッケル、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭのｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３１】
実施例６９
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ塩化コバルト、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭのｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３２】
実施例７０
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸亜鉛、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭのｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３３】
実施例７１
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ酢酸銅、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭのｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３４】
実施例７２
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの５０ｍＭトリス・ＨＣｌ（ｐＨ８．５）に溶解後、終濃度４０ｍＭになるようにシステアミンを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３５】
実施例７３
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭのトリレン−３，４−ジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３６】
実施例７４
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭの４−クロロ−ｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３７】
実施例７５
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭの３，４−ジアミノ安息香酸を含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３８】
実施例７６
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの４Ｍ尿素に溶解し、４Ｍ酢酸ナトリウム、２０ｍＭ酢酸、０．４Ｍグリオキシル酸、２０ｍＭ硫酸銅、４Ｍ尿素溶液１ｍｌと混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭの２，３−ジアミノフェノールを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１３９】
実施例７７
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの３Ｍ尿素−２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）に溶解し、１Ｍイミダゾール、０．５Ｍグリオキシル酸、２０ｍＭ硫酸銅、２．５Ｍ尿素溶液１ｍｌとを混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭ ｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１４０】
実施例７８
参考例１１で得た、Ｎ末端にメチオニンの付加したＭｅｔ−ＰＴＨ（１−３４）５ｍｇを１ｍｌの３Ｍ尿素−２０ｍＭトリス／ＨＣｌ（ｐＨ８．０）に溶解し、４Ｍ酢酸ナトリウム、０．８Ｍ酢酸、０．４Ｍグリオキシル酸、１０ｍＭ硫酸銅、２．５Ｍ尿素溶液１ｍｌとを混合して、室温で１時間反応した。反応終了後、遠心分離を行い、その上清を実施例６６と同様にＯＤＰ−５０カラム用いた高速液体クロマトグラフィーにより精製し、Ｍｅｔ−ＰＴＨ（１−３４）のジケトン体画分を得た。
【０１４１】
次にこの画分を凍結乾燥し、４ｍｌの蒸留水に溶解後、８０ｍＭ ｏ−フェニレンジアミンを含む、４Ｍ酢酸、４Ｍ酢酸ナトリウムの溶液４ｍｌを加え、脱気窒素ガスシールを行った後、３７℃で１７時間反応した。反応終了後、実施例６６と同じ方法により精製し、ＰＴＨ（１−３４）の凍結乾燥粉末を得た。
【０１４２】
【発明の効果】
本発明により、Ｎ末端に酸化されていてもよいメチオニン残基を有するペプチド、蛋白質またはその塩から、該メチオニン残基のみを選択特異的かつ効率的に取り除くことができる。また、本発明の方法によれば、ペプチドまたは蛋白質の種類に拘わらず、しかもマイルドな条件下でＮ末端のメチオニン残基を化学的に除去することができるので、遺伝子工学的手法により製造されたメチオニンの付加した蛋白質を原料にして、天然型のアミノ酸配列を有する蛋白質を工業的に有利に製造することができる。
【０１４３】
【配列表】

【０１４４】

【０１４５】

【０１４６】

【図面の簡単な説明】
【図１】実施例２ａ) で得られた電気泳動の結果を示す。
【図２】実施例５ａ) で得られた電気泳動の結果を示す。
【図３】実施例７ａ) で得られた電気泳動の結果を示す。
【図４】実施例９ａ) で得られた電気泳動の結果を示す。
【図５】実施例１１ａ) で得られた電気泳動の結果を示す。
【図６】実施例２４ａ) で得られた電気泳動の結果を示す。
【図７】実施例３８ａ) で得られた電気泳動の結果を示す。
【図８】実施例６７ａ) で得られた電気泳動の結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for removing methionine from peptides (including proteins) having methionine at the N-terminus or salts thereof.
[0002]
[Prior art]
It is known that when a protein is biosynthesized in a cell, its N-terminus begins with methionine corresponding to the start codon AUG of mRNA. However, since this methionine is removed by subsequent processing, it is usually no longer present in the finished mature protein molecule.
Advances in gene recombination technology have made it possible to produce useful proteins using microorganisms and animal cells, such as E. coli, but the methionine remains in the proteins produced by this method. Has been found. For example, in human growth hormone expressed in E. coli, the methionine addition rate is almost 100% [Nature,293, 408 (1981)], 50% for interferon-α [J. Interferon Res.1, 381 (1981)], in the case of non-glycosylated human interleukin-2, in addition to the molecular species beginning with alanine (rIL-2) as in the case of natural human interleukin-2, the molecular species in which methionine is further added to the amino terminus ( The presence of Met-rIL-2) has been observed.
On the other hand, as a method of chemically removing the amino acid at the N-terminal, Dixon in 1964 caused DL-alanylglycine to react with glyoxylic acid, pyridine, and copper acetate to cause an amino group transfer reaction. [Biochemistry Journal (Biochem. J),92, 661 (1964)], and further reported that when a compound is reacted with thiosemicarbazide, cleavage of the amide bond occurs to produce glycine [Biochemistry Journal (Biochem. J),90, 2C (1964)]. Subsequently, this reaction was applied to cytochrome C-551 (Pseudomonas cytochrome c-551), and it was reported that N-terminal glutamic acid was removed [Biochemistry Journal (Biochem. J),94, 463 (1965)]. However, these are limited to the removal of the N-terminal amino acid of the synthetic peptide or mature protein, and no example of effectively utilizing this reaction for removing the methionine added to the N-terminus of the recombinant protein can be found.
[0003]
[Problems to be solved by the invention]
Even for the same protein, there is a possibility that the higher molecular structure, biological activity, and stability of the molecular species with methionine added at the N-terminus and those without the methionine are different from each other. It is believed that the addition may result in increased antigenicity. Therefore, it is considered to be meaningful to establish an N-terminal methionine removal method corresponding to this start codon from the viewpoint of industrial use.
In order to solve this problem, a method for removing methionine by decomposition of cyanogen bromide (BrCN) was proposed [Science,198, 1056 (1977)], but in this case, it is premised that there is no methionine residue in the desired mature protein, and depending on the method of subjecting the protein to a harsh chemical reaction, the result is never satisfactory. Cannot be obtained.
There is no known chemical method for selectively and efficiently removing a methionine residue at the N-terminus from a peptide or protein having a methionine residue at the N-terminus, regardless of the type of peptide or protein. Although not, this is believed to be due to the difficulty in finding a chemical reaction that can remove the N-terminal methionine under mild conditions without denaturing the final product peptide or protein. . In particular, when removing methionine added at the N-terminus from a protein having a relatively large molecular weight and produced by genetic engineering, particularly a protein intended for use as a pharmaceutical, the activity of the protein after methionine removal In general, it is necessary to allow the reaction to proceed without heating in a weakly acidic to weakly alkaline aqueous solution, and there are many chemical reaction conditions. The current situation is that we cannot find out.
[0004]
[Means for Solving the Problems]
The present inventors have intensively studied to provide a method for producing a protein having a natural amino acid sequence by cleaving only the N-terminal methionine in a protein produced by genetic engineering. After converting methionine into an α-diketone in the protein, the result of diligent efforts to find a method for removing the N-terminal methionine from the methionine-added protein by the action of organic diamines, is represented by the following formula (I). The peptide or protein to which methionine is added is reacted with glyoxylic acid as an α-diketone, copper sulfate that can donate a transition metal ion, and pyridine as an amine to perform an amino group transfer reaction to convert methionine into α- After conversion to diketone, it is further hydrolyzed by reaction with o-phenylenediamine, which is a diamine. To find a method for removing a N-terminal methionine from a peptide or protein with methionine added to obtain a peptide or protein without a methionine added to the N-terminal without reducing its activity. The present invention has been completed.
[Chemical 1]

[In the formula (I), X may be any amino acid residue or a peptide chain having an arbitrary number of amino acids of 2 or more, but from a practical aspect, X is a protein chain produced by genetic engineering. A corresponding part of the peptide chain can be mentioned. In the present specification, when referred to as a protein or peptide, the peptide or protein comprising a plurality of amino acids may be any of a non-glycosylated or glycosylated peptide or protein. ]
[0005]
That is, the present invention
(1) A method for removing the methionine residue, comprising reacting a peptide having a methionine residue which may be oxidized at the N-terminus or a salt thereof with an α-diketone, followed by hydrolysis.
(2) The method according to (1) above, wherein the peptide having a methionine residue which may be oxidized at the N-terminus is a peptide produced by genetic engineering;
(3) The aforementioned peptide produced by genetic engineering is a growth hormone, neurotrophin-3, betacellulin, parathyroid hormone or interleukin-2 to which a methionine residue which may be oxidized at the N-terminus is added (2) The method according to the description;
(4) The method according to (1) above, wherein an α-diketone is reacted in the presence of a transition metal ion;
(5) The method according to (1) above, wherein the α-diketone is reacted in the presence of a base;
(6) The method according to (1), wherein the α-diketone is reacted in the presence of a transition metal ion and a base;
(7) The method according to (1) above, wherein the α-diketone is glyoxylic acid or a salt thereof;
(8) The method according to (4) above, wherein the transition metal ion is a copper ion;
(9) The method according to (5) above, wherein the base is pyridine;
(10) The method according to (1) above, wherein hydrolysis is performed using a base;
(11) The method according to (10) above, wherein the base is an amine;
(12) The method according to (10) above, wherein the base is a diamine or thio or selenosemicarbazide;
(13) The method according to (12) above, wherein the diamine is o-phenylenediamine;
(14) Human growth hormone or a salt thereof, which is produced by genetic engineering and added with methionine at the N-terminus, and glyoxylic acid or a salt thereof are reacted in the presence of copper sulfate and pyridine, and then reacted with o-phenylenediamine. A method for producing human growth hormone or a salt thereof,
(15) After reacting neurotrophin-3 or a salt thereof, which is produced by genetic engineering and having methionine added to the N-terminus, with glyoxylic acid or a salt thereof in the presence of copper sulfate and pyridine, o-phenylenediamine A method for producing neurotrophin-3 or a salt thereof, characterized by reacting with
(16) Human betacellulin or a salt thereof produced by genetic engineering and having methionine added to the N-terminus is reacted with glyoxylic acid or a salt thereof in the presence of copper sulfate and pyridine, and then reacted with o-phenylenediamine. A method for producing human betacellulin or a salt thereof,
(17) Human interleukin-2 or a salt thereof produced by genetic engineering and having methionine added to the N-terminus thereof and glyoxylic acid or a salt thereof are reacted in the presence of copper sulfate and pyridine, and then o-phenylenediamine. A method for producing human interleukin-2 or a salt thereof, characterized by reacting with
(18) A parathyroid hormone produced by genetic engineering and methionine added to methionine or a salt thereof and glyoxylic acid or a salt thereof are reacted in the presence of copper sulfate and pyridine, and then reacted with o-phenylenediamine. A method for producing parathyroid hormone or a salt thereof,
(19) Formula CH_Three-S (O)_m-(CH₂)₂-CO-CO-X [wherein m represents an integer of 0 to 2, and X represents an amino acid residue or a peptide chain. Or a salt thereof; and
(20) A method for producing an amino acid, peptide or salt thereof, which comprises hydrolyzing the compound described in (19).
[0006]
In this specification, the methionine residue which may be oxidized indicates a methionine residue or an S oxidized form thereof, and examples of the S oxidized form of the methionine residue include a sulfoxide and a sulfone form.
Peptides or proteins having a methionine residue which may be oxidized at the N-terminus include the formula CH_Three-S (O)_m-(CH₂)₂-CH (NH₂) -CO-X [wherein m represents an integer of 0 to 2, and X represents an amino acid residue or a peptide chain. ] These may form a salt, and any salt may be used as long as it does not inhibit the reaction of the present invention, and among them, a pharmaceutically acceptable salt. And salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, phthalic acid, fumaric acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfone Examples thereof include salts with organic acids such as acids, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts, and ammonium salts.
[0007]
In the above formula, m is preferably 0. X is preferably a peptide chain having 2 or more amino acids.
The peptide used in the removal method of the present invention may be a so-called peptide having less than 50 amino acids or a so-called protein having 50 or more amino acids.
Thus, in the present specification, the term “peptide” includes not only molecules having 50 or less amino acids but also molecules having 50 or more amino acids. Among them, the number of amino acids is 50 or more. These molecules (so-called proteins) are preferably used.
Preferable peptides include peptides having 2 to 1000 amino acids, more preferably peptides having 15 to 500 amino acids, and specific examples thereof include growth hormone (GH), parathyroid hormone (PTH), Insulin, nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor, neurotrophin-3, 4 or 6, central nerve growth factor, glial growth factor, lung-derived neurotrophic factor, Epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor α or β, vascular endothelial cell growth factor, tissue plasminogen activator, urokinase, protein C, thrombomodulin, osteogenic factor, calcitonin , Insulin-like growth factor, interferon -Α, β or γ, interleukin-1 (α, β) -12, granule colony stimulating factor, granule macrophage colony stimulating factor, granule macrophage stimulating factor, thrombopoietin, erythropoietin, PACAP, atrial natriuretic peptide, Endothelin, megakaryocyte growth factor, blood stem cell growth factor, hepatocyte growth factor, motilin, immunotoxin, tumor necrosis factor, hirudin, corticotropin, angiotensin, angiotensin 2 and its peptide antagonist, angiotensin 3, bradykinins, bradykinin enhancing factor , Α, β or γ endorphin, enkephalin, chemotactic factor for neutrophils, gastrin, glucagon, growth hormone releasing factor, kyotorphin, kallidin, gonadotropin releasing hormone, mast cell degranulating peptide, Melanocyte stimulating hormone, neurotensin, trypsin inhibitor, oxytocin, proinsulin C-peptide, secretin, somatostatin, thyroid stimulating hormone releasing hormone, ubiquitin, urogastron, vasopressin, kinins, tuftsin, somatomedin, corticotropin releasing factor, insulin-like growth Factor, calcitonin gene-related peptide, PTHrP, VIP, DHI, insulinotropin, GRP, CCK-PZ, Galanin (Galanin), Acatrum peptide (Antrum Peptide), PPY, Pancreatic Polypeptide, PSP, Pancreatastatin, hCG, hCS Relaxin, serum thymus factor, thymopoietin, thymosin, factor XIII, factor VIII, prourokinase, SOD, factor VIIa, antithrombin Proteins and their muteins (those with one or more amino acid substitutions, deletions or additions to natural proteins and exhibiting biological or immunological activity equal to or higher than that of natural proteins), or chemical Examples include known or novel peptides produced by synthesis, etc. Among them, proteins produced by genetic engineering, especially methionine produced by genetic engineering and optionally oxidized at the N-terminus are added. Growth hormone, neurotrophin-3, betacellulin, parathyroid hormone, interleukin-2 and the like are preferably used.
The natural protein described above may be derived from any animal species, but for practical use, a human-derived protein is preferably used.
[0008]
  In the present specification, α-JikeTons may be any as long as they can promote the amino group transfer reaction of the above-described peptides or salts thereof, for example, the formula R¹-CO-CO-R²[Wherein R¹Represents a lower alkyl or phenyl group (preferably hydrogen or methyl, more preferably hydrogen) optionally substituted with hydrogen or a carboxyl group, and R²Represents a hydroxyl group, a lower alkoxy group or an amino group (preferably a hydroxyl group) which may be substituted with lower alkyl. Or a salt thereof and the like. In the above formula, R¹Examples of the lower alkyl group represented by the formula include alkyl groups having about 1 to 6 carbon atoms such as methyl, ethyl, propyl, i-propyl, butyl, i-butyl, sec-butyl, t-butyl, etc.²Examples of the lower alkoxy group represented by the formula include alkoxy groups having about 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, i-propoxy, butoxy, i-butoxy, sec-butoxy and t-butoxy. R²As the amino group optionally substituted by lower alkyl represented by¹And an amino group optionally having 1 to 2 lower alkyl groups represented by the formula: Furthermore, examples of the salt include the same salts as those described above for peptides or proteins. α-JikeSpecific examples of tons include glyoxylic acid, pyruvic acid, oxalacetic acid, phenylglyoxylic acid, 2-oxoglutaric acid, etc. Among them, glyoxylic acid is preferably used.
[0009]
The transamination reaction between a peptide having a methionine residue optionally oxidized at the N-terminal or a salt thereof and an α-diketone is usually 1 to 10,000 moles (preferably 1 mole of the peptide or salt thereof). It is preferable to react α-diketone of about 2000 to 4000 mol) at about 0 to 70 ° C. (preferably about 20 to 40 ° C.) for about 5 minutes to 2 hours (preferably about 15 minutes to 1 hour). . Any buffer solution (eg, phosphate buffer solution, acetate buffer solution, citrate buffer solution, etc.) may be used as long as it does not inhibit the above-mentioned transamination reaction. Among them, an acetate buffer solution is preferable. Used. In addition, the pH of the reaction is adjusted to about 2 to 9, in particular, about 4 to 7, particularly about 5 to 6, and a peptide having a methionine residue which may be oxidized at the N-terminus or a salt thereof The reaction should be allowed to proceed under conditions that do not denature.
In order to promote the transamination reaction, it is preferable to react the α-diketone in the presence of a transition metal ion, and usually 0.001 to 0.1 mol (preferably with respect to 1 mol of the α-diketone). Is preferably about 0.01 to 0.05 mol) of transition metal ions. Examples of transition metal ions include copper ions (Cu⁺, Cu²⁺), Cobalt ion (Co²⁺, Co³⁺), Nickel ion (Ni²⁺, Ni³⁺), Iron ion (Fe²⁺, Fe³⁺), Zinc ion (Zn²⁺), Aluminum ion (Al³⁺), Manganese ions (Mn²⁺Etc.), gallium ion (Ga³⁺), Indium ions (In³⁺), Magnesium ion (Mg²⁺), Calcium ion (Ca²⁺In particular, copper ions, cobalt ions, etc., especially copper ions (Cu²⁺) Is preferably used. These transition metal ions are usually added to the reaction solvent as a salt with an inorganic acid such as sulfuric acid, nitric acid, hydrochloric acid or perchloric acid or as a salt with an organic acid such as acetic acid, oxalic acid, citric acid or carbonic acid. Among these, copper sulfate, copper acetate, especially copper sulfate is preferably used.
[0010]
The α-diketone is preferably reacted in the presence of a base, and usually about 0.1 to 2 mol (preferably 0.5 to 1.0 mol) per 1 mol of the α-diketone. It is preferred to use a base. Examples of the base include alkylamines such as triethylamine and tributylamine, aromatic amines such as N, N-dimethylaniline, pyridine, lutidine, collidine, 4- (dimethylamino) pyridine and imidazole, and organic bases such as urea. Among them, aromatic amines, particularly pyridine is preferably used.
[0011]
Further, in the above-mentioned transamination reaction, α-diketone is preferably reacted in the presence of a transition metal ion and a base. Practically, three components (for example, transition metal ion, base and α-diketone) are used. , Copper sulfate, pyridine, glyoxylic acid, etc.) is added to an aqueous solution containing a peptide having a methionine residue which may be oxidized at the N-terminus or a salt thereof, and the transamination reaction proceeds. Let
Obtained by the transamination reaction and having the formula CH_Three-S (O)_m-(CH₂)₂-CO-CO-X [wherein m represents an integer of 0 to 2, and X represents an amino acid residue or a peptide chain. Or a salt thereof is a novel compound, and is isolated and purified from the reaction solution by known purification means of peptides or proteins, such as extraction, salting out, partitioning, recrystallization, chromatography, etc. It can also be subjected to the next hydrolysis reaction as it is.
The diketone obtained by the transamination reaction is usually subjected to a hydrolysis reaction with a base to convert it to an amino acid, peptide or salt thereof from which the N-terminal optionally oxidized methionine residue has been removed. Can do.
[0012]
Examples of the base used for the hydrolysis reaction include alkylamines such as cysteamine, triethylamine and tributylamine, aromatic amines such as N, N-dimethylaniline, pyridine, lutidine, collidine, 4- (dimethylamino) pyridine and imidazole. , Diamines such as o-phenylenediamine, tolylene-3,4-diamine, 3,4-diaminobenzoic acid, 2,3-diaminophenol, 4-chloro-o-phenylenediamine (preferably aromatic diamines, Among them, o-phenylenediamines), thiosemicarbazides such as thiosemicarbazide, acetone thiosemicarbazide and phenylthiosemicarbazide, and amines such as selenosemicarbazide such as selenosemicarbazide and acetone selenosemicarbazide can be used. But it is, among others, amines, especially diamines or thiosemicarbazide are preferred, especially, o- phenylenediamine is preferably used.
[0013]
The amount of the base is usually about 10,000 to 10,000 moles, preferably about 500 to 2,000 moles per mole of the diketone body. The hydrolysis reaction is usually preferably allowed to proceed at about 0 to 70 ° C. (preferably about 20 to 40 ° C.) for about 1 to 50 hours (preferably about 10 to 25 hours). In the reaction, a buffer solution is preferably used as a solvent, and examples of the buffer solution include a phosphate buffer solution, an acetate buffer solution, and a citrate buffer solution. Any buffer may be used as long as it does not inhibit the hydrolysis reaction described above, and among them, an acetate buffer is preferably used. The reaction pH is adjusted to about 2 to 9, especially about 3 to 7, especially about 4 to 6 neutral, and the reaction is performed under conditions where the resulting amino acid, peptide or salt thereof is not denatured. It is better to proceed.
The amino acid, peptide or salt thereof thus obtained can be isolated and purified from the reaction solution by known purification means such as extraction, salting out, partitioning, recrystallization, chromatography, etc., but it is preferable. Examples include purification methods such as ion exchange chromatography through SP-Sepharose (Pharmacia Biotech Co., Ltd.) or DEAE-5PW (Tosoh Corp.).
[0014]
The polypeptide produced according to the present invention does not have a methionine at its N-terminus, and is obtained as having the same amino acid sequence as a natural physiologically active polypeptide, and therefore has the same activity as a natural polypeptide. It can be used safely as a pharmaceutical or diagnostic agent with low toxicity.
According to the present invention, the N-terminal methionine can be specifically removed from the methionine-added peptide.
In the description of the present invention and [drawings], the abbreviations such as amino acids are based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are shown below. When there is optical isomerism with respect to amino acids, L-form is indicated unless otherwise specified.
[0015]
SDS: sodium dodecyl sulfate
Gly: Glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: Cysteine
Met: Methionine
Glu: Glutamic acid
Gln: Glutamine
Asp: Aspartic acid
Asn: Asparagine
Lys: Lysine
Arg: Arginine
His: histidine
Phe: Phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asx: Asp + Asn
Glx: Glu + Gln
[0016]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described more specifically with reference to the following reference examples and examples, but the present invention is not limited thereto.
【Example】
Reference example 1
Production of Met-rhGH:
Met-rhGH was produced according to the method described in Reference Example 4 of JP-A-62-1171699.
(I) Producing bacteria
A transformant Escherichia coli K12 χ1776 / pHGH 107 (ATCC 31538) having a human growth hormone gene distributed by ATCC was used.
(Ii) Culture
Transformant Escherichia coli K12 χ1776 / pHGH 107 (ATCC 31538, IFO 14505) in 2 L volume Mayer's Bacto tryptone 1%, Bacto yeast 0.5%, sodium chloride 0.5%, tetracycline / hydrochloric acid 10 mg / L, Inoculated into 1 L of liquid medium (pH 7.0) containing ampicillin sodium 10 mg / L, thymine 20 mg / L and diaminopimelic acid 100 mg / L, and cultured with shaking at 37 ° C. overnight. This culture broth was made up of sodium monohydrogen phosphate 1.68%, potassium dihydrogen phosphate 0.30%, ammonium chloride 0.10%, sodium chloride 0.05%, antifoaming agent 200 mg / L, glucose 1.00% , Casamino acid 1.00%, magnesium sulfate 246 mg / L, tetracycline / hydrochloric acid 10 mg / L, ampicillin sodium 10 mg / L, thymine 20 mg / L and diaminopimelic acid 100 mg / L The mixture was transferred to a 50 L jar fermenter, and cultured with aeration and stirring at 37 ° C. for 10 hours. The culture broth was centrifuged to collect microbial cells.
[0017]
Reference example 2
Take out 2 kg of wet cells obtained in Reference Example 1, add 6 L of 50 mM Tris-HCl buffer (pH 8.0) containing 8 M guanidine hydrochloride to the cells, and stir well until the cells no longer clump together. After dissolution, centrifugation (10,000 rpm, 60 minutes) was performed to obtain about 6 L of a cell extract. This bacterial cell extract is dialyzed twice against about 70 L of 10 mM Tris-HCl buffer (pH 7.0), and about 9 L of the dialysis internal solution obtained after the completion of dialysis is adjusted to a final concentration of 20% saturated ammonium sulfate. Then, ammonium sulfate was added and dissolved, and then centrifuged (4,200 rpm, 60 minutes) to obtain about 10 L of a centrifugal supernatant. The supernatant was divided into two portions, passed through a phenyl-Toyopearl 650C column (5 cmφ × 50 cm), adsorbed and washed, and (1) 10 mM Tris-HCl buffer (pH 7.0) containing 40% saturated ammonium sulfate. ) And {circle around (2)} by concentration gradient elution with 10 mM Tris-HCl buffer (pH 7.0), and the Met-rhGH fraction was collected and dialyzed against 50 mM sodium bicarbonate solution (pH 8.2). The obtained dialyzed solution was centrifuged (4,200 rpm, 45 minutes) to obtain about 3.7 L of a centrifugal supernatant. This supernatant was divided into four portions and passed through a DEAE-Toyopearl 650M column (4 cmφ × 50 cm), adsorbed and washed, and then, (1) 10 mM sodium hydrogen carbonate solution (pH 8.2) and (2) 1M. Met-rhGH was eluted by concentration gradient elution with 10 mM sodium hydrogen carbonate solution (pH 8.2) containing sodium chloride, and the collected Met-rhGH fraction was added to 50 mM sodium hydrogen carbonate solution (pH 8.2). And dialyzed. About 1.2 L of the obtained dialyzed internal solution was added to a DEAE-5PW column (55 mmφ × 20 cm, Tosoh) and (1) 10 mM sodium hydrogen carbonate solution (pH 8.2) and (2) 10 mM hydrogen carbonate containing 1 M sodium chloride. Adsorption and elution were performed by high-performance liquid chromatography using a concentration gradient elution method with a sodium solution (pH 8.2) to obtain about 600 ml of a Met-rhGH fraction. After concentrating this solution with Amicon Diaflow (YM-10 membrane, 76 mmφ, Amicon), about 294 ml of this concentrated solution was equilibrated with 100 mM sodium hydrogen carbonate solution (pH 8.2) (Toyopearl HW-50F column ( Gel filtration was performed using 8 cmφ × 50 cm) to obtain a Met-rhGH fraction. Subsequently, the mixture was filtered through a Milex GV filter (0.22 μm, Millipore) to obtain 869 mg of Met-rhGH.
[0018]
Example 1
50 mg of human growth hormone (Met-rhGH) added with methionine at the N-terminus obtained in Reference Example 2 was lyophilized, dissolved in 40 ml of 50 mM phosphate buffer (pH 8.0), and then 0.1 M sulfuric acid. A mixed solution of 5 ml of copper, 2.3 g of glyoxylic acid and 5 ml of pyridine was added and reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2 M acetic acid-2 M sodium acetate solution, and the solution used for equilibration was flowed at a flow rate of 6 ml / min. The diketone fractions of Met-rhGH were pooled. Subsequently, o-phenylenediamine was added to this fraction so as to have a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 20 hours. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM Tris-HCl buffer (pH 8.0), and the buffer used for equilibration was 6 ml / min. Development was carried out at a flow rate, and human growth hormone (rhGH) fractions with no methionine added at the N-terminus were pooled. The pooled rhGH fraction was adsorbed on DEAE-5PW (21.5 mm ID × 150 mm L) equilibrated with 20 mM Tris-HCl buffer (pH 8.0), and then 0-100% B (B = 20 mM Tris-HCl buffer + 1 M). Elution was carried out with a step gradient of NaCl, pH 8.0) for 30 minutes at a flow rate of 8.5 ml / min, and the rhGH fractions were pooled. Furthermore, after the rhGH fraction was passed through Toyopearl HW-50 (20 mm ID × 600 mm L) (Tosoh Corporation) equilibrated with 5% ethanol, the solution used for equilibration was developed at a flow rate of 6 ml / min. The rhGH fractions were pooled and then lyophilized to obtain rhGH lyophilized powder.
[0019]
Example 2
(Characteristic determination of rhGH)
a) Analysis using SDS polyacrylamide gel electrophoresis
The rhGH obtained in Example 1 was sample buffer [Laemmli, Nature,227, 680 (1970)], heated with 100 mM DTT at 100 ° C. for 1 minute, and then electrophoresed with Multigel 10/20 (Daiichi Chemical Co., Ltd.). When the gel after electrophoresis was stained with Coomassie brilliant blue, a single band of protein was observed, and the purified product was almost single. The results are shown in FIG. In FIG. 1, Lanes 1-3 represent rhGH (3 μg), blank and molecular weight markers.
b) N-terminal amino acid sequence analysis
The N-terminal amino acid sequence of rhGH obtained in Example 1 was determined using a gas phase protein sequencer (Applied Biosystems model 477A). As a result, the N-terminal amino acid sequence of the obtained rhGH coincided with the N-terminal amino acid sequence of rhGH deduced from the cDNA base sequence. The results are shown in [Table 1].
[Table 1]

[0020]
c) Amino acid composition analysis
About 20 μg of rhGH obtained in Example 1 was used, and its amino acid composition was determined by an amino acid analyzer (Beckman System 6300E). As a result, the amino acid composition was inferred from the nucleotide sequence of the rhGH cDNA. The results are shown in [Table 2].
[Table 2]

d) C-terminal amino acid analysis
Using 15 nmol of rhGH obtained in Example 1, the C-terminal amino acid was determined by an amino acid analyzer (Beckman System 6300E). It coincided with the C-terminal amino acid deduced from the nucleotide sequence of rhGH cDNA. The results are shown in [Table 3].
[Table 3]

[0021]
Example 3
(Measurement of rhGH activity)
“Journal of Clinical Endocrinology & Metabolism” using Nb 2 cells,51, 1058 (1980) ", the purified rhGH product obtained in Example 1 had almost the same activity as that of the standard product.
[0022]
Reference example 3
Production of Met-NT-3:
Met-NT-3 was produced according to the methods described in Reference Examples 1 to 3 and Examples 1-2 of Japanese Patent Application No. 8-74775.
(1) Cloning of NT-3 DNA
After infecting E. coli Y1090 with a λgt 11 cDNA library derived from human glioma (Clontech Laboratories, Inc.), about 6 × 10^FiveEach phage was seeded in NZCY medium (described in Molecular Cloning, A Laboratory Manual, Clod Spring Harbor Laboratory, 1982) and cultured at 37 ° C. for 5 hours. Next, the nylon membrane was placed on the plate, left for 1 minute, and then removed from the plate. The nylon membrane is immersed in 0.5M NaOH-1.5M NaCl, then 1.5M NaCl-0.5M Tris-HCl pH 8.0, and further immersed in 2 × SSC (see Molecular Cloning, A Laboratory Manual, supra) and air-dried. Then, it was left at 80 ° C. for 2 hours.
Human βNGF [Nature,303, 821 (1983)] is chemically synthesized and [α-³²Probes were made by labeling with P] dCTP.
Hybridization was performed according to the method described in Molecular Cloning, A Laboratory Manual, Clod Spring Harbor Laboratory, 1982, using the nylon membrane and probe obtained above. That is, the nylon membrane was immersed in a hybridization solution containing the probe and kept at 65 ° C. for 16 hours. The nylon membrane was washed with 2 × SSC-0.1% SDS at room temperature and then washed with 1 × SSC-0.1% SDS at 60 ° C. Then positive clones were obtained by autoradiography. CDNA was excised from the thus obtained clone λβGN1321 with EcoRI and inserted into the EcoRI site of plasmid pUC118 (Takara Shuzo Co., Ltd.) to obtain plasmid pUNK5.
[0023]
(2) Construction of NT-3 expression vector for E. coli
The NT-3 cDNA inserted in the plasmid pUNK5 obtained in Reference Example 3 (1) has a ScaI site near the region encoding the 11th tyrosine residue at the N-terminus of NT-3. An NsiI site is present in the vicinity of 50 bases downstream of the stop codon. Therefore, a 0.3 kb ScaI-NsiI fragment was isolated from pUNK5, and adapters NGFTE-1 (35 mer), NGFTE-2 (33 mer), NGFTE-3 (7 mer) and NGFTE-4 (15 mer) were ligated with T4 DNA ligase. Thereafter, the fragments were treated with restriction enzymes NdeI and BamHI to obtain a 0.3 kb NdeI-BamHI fragment.
The adapter is shown below.
NGFTE-1: 5 'TATGTACGCGGAGCATAAGAGTCACCGAGGGGAGT 3' 35mer (SEQ ID NO: 1)
NGFTE-2: 5'ACTCCCCTCGGTGACTCTTATGCTCCGCGTACA 3 '33mer (SEQ ID NO: 2)
NGFTE-3: 5'TGCCAGG 3 '7mer
NGFTE-4: 5'GATCCCTGGCATGCA 3 '15mer (SEQ ID NO: 3)
On the other hand, expression vector pET-3C having T7 promoter [Rosenberg et al., Gene,56, 125 (1987)] was digested with NdeI and BamHI, and a 4.4 kb NdeI-BamHI fragment was isolated.
The 4.4 kb NdeI-BamHI fragment and the 0.3 kb NdeI-BamHI fragment obtained above were ligated with T4 DNA ligase, then introduced into Escherichia coli DH1, and the resulting ampicillin resistant transformant [Escherichia coli DH1 / pENGFT103 The plasmid isolated from the above was named pENGFT103.
[0024]
(3) Production of Met-NT-3 for E. coli
Using the NT-3 expression vector pENGFT103 and the T7 lysozyme expression vector pLysS obtained in Reference Example 3 (2), Escherichia coli MM294 (DE3) [Molecular Endocrinology,4, 869 (1990)] to obtain a transformant E. coli MM294 (DE3) / pLysS, pENGFT103 (IFO 15932, FERM BP-5383).
Transformant E. coli MM294 (DE3) / pLysS, pENGFT103 LB medium containing 50 μg / ml ampicillin and 15 μg / ml chloramphenicol [1% peptone, 0.5% yeast extract, 0.5% chloride Sodium] In a 2 liter flask containing 1 liter, the mixture was cultured with shaking at 30 ° C. for 8 hours. The obtained culture broth was added to 20 liters of main fermentation medium [1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.05. To 50 liter fermenter charged with 2% magnesium sulfate, 0.02% antifoam, 0.0025% ferrous sulfate, 0.0005% thiamine hydrochloride, 1.5% glucose, 1.5% casamino acid) After transplanting, aeration and agitation culture was started at 30 ° C. When the turbidity of the culture reached about 500 kret units, 100 mg / L of isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was further continued. After 7 hours, the culture was terminated. did. By centrifuging this culture-terminated solution, about 340 g of wet cells were obtained and stored frozen at -80 ° C.
[0025]
(4) Production of Met-NT-3 for E. coli (mass production)
LB medium containing 50 μg / ml ampicillin and 15 μg / ml chloramphenicol in the above transformant E. coli MM294 (DE3) / pLysS, pENGFT103 [1% peptone, 0.5% yeast extract, 0.5% Sodium chloride] was cultured with shaking in a 2 liter flask containing 1 liter at 30 ° C. for 16.5 hours. The obtained culture broth was transplanted into a 50 liter fermenter containing 20 liters of LB medium (containing 0.02% antifoaming agent, 50 μg / ml ampicillin and 15 μg / ml chloramphenicol), The culture was aerated and stirred at 30 ° C. for 7 hours. This culture solution was added to 360 liters of the main fermentation medium [1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.05% sulfuric acid. Transplanted into a 500 liter fermenter containing magnesium, 0.02% antifoam, 0.0025% ferrous sulfate, 0.0005% thiamine hydrochloride, 1.5% glucose, 1.5% casamino acid. Then, aerated stirring culture was started at 30 ° C. When the turbidity of the culture solution reaches about 500 kret units, 100 mg / L of isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was further continued. Was centrifuged to obtain about 6 kg of wet cells and stored frozen at -80 ° C.
[0026]
(5) Activation of Met-NT-3
40 g of the wet microbial cells obtained in Reference Example 3 (3) was taken out and suspended by adding 240 ml of 10 mM EDTA (pH 7.0) to the microbial cells, and then Sonifier 450 (Branson) was cooled under ice cooling. The cells were disrupted using ultrasonic waves and centrifuged (10000 rpm, 1 hour). The obtained pellet was washed by performing the same operation twice. Next, 160 ml of 50 mM Tris-HCl / 4 M urea / 5 mM dithiothreitol (DTT) (pH 8.0) was added and homogenized, followed by centrifugation. The resulting pellet was added with 20 mM citric acid / 8 M urea (pH 3.0). After dissolution by adding 120 mM, the supernatant is separated from the sediment, and the same operation is again performed on the sediment to mix the supernatant obtained earlier and the supernatant obtained earlier. , 240 ml of pellet solution was obtained.
The solution was diluted by adding 760 ml of a 100 mM acetic acid solution, passed through a Sephadex G-25 column (11.3 cmφ × 50) equilibrated with 100 mM acetic acid, and the modified Met-NT-3 solution from which urea was removed. 1640 ml were obtained. This solution was allowed to stand at 4 ° C. for 2 days, and then 50 mM phosphate buffer / 12.5% sucrose (pH 6.8) was added to make 8.5 L. Then, the mixture was allowed to stand again at 4 ° C. for 2 days for activation. After standing, 200 mM copper sulfate was added so as to have a final concentration of 10 μM, and after stirring, the mixture was allowed to stand again at 4 ° C. for 2 days in order to continue activation.
[0027]
(6) Purification of Met-NT-3
The solution after the activation in Reference Example 3 (5) was added to 100 mM phosphate buffer / 0.1% 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS) (pH 6. 0) equilibrated with SP-Sepharose Fast Flow column (2.5 cmφ × 12 cm, Pharmacia Biotech), and after adsorption, 200 ml of 100 mM phosphate buffer / 0.1% CHAPS / 200 mM sodium chloride (pH 6. The column was washed with 0), and then eluted with 100 mM phosphate buffer / 0.1% CHAPS / 400 mM sodium chloride (pH 6.0) to obtain an eluate containing Met-NT-3. After this eluate was adsorbed on a Resource 15 RPC column (2 cmφ × 30 cm, Pharmacia Biotech), (1) concentration elution with 16% acetonitrile / 0.1% TFA and (2) 36% acetonitrile / 0.1% TFA The eluate was freeze-dried to obtain about 28 mg of Met-NT-3 white powder.
[0028]
Example 4
To a solution prepared by dissolving 50 mg of Met-NT-3 obtained in Reference Example 3 (6) with 4.6 ml of distilled water, a solution prepared by mixing 92.5 mg of glyoxylic acid, 200 μl of pyridine, and 200 μl of 100 mM copper sulfate solution was added. After gently stirring, the mixture was allowed to react at room temperature for 15 minutes. After completion of the reaction, this reaction solution was passed through a Sephadex G-25 column (2.5 cmφ × 59 cm) equilibrated with 2M acetate buffer (pH 4.9) to collect 36 ml of Met-NT-3 diketone body. It was. After adding 78 mg of o-phenylenediamine to this solution and reacting at 37 ° C. for 15 hours, the reaction-terminated solution was equilibrated with 2M acetic acid buffer (pH 4.9) using a Sephadex G-25 column (2. 5 cmφ × 59 cm), and 75 ml of the obtained NT-3 fraction was concentrated with (1) 0.1% TFA and (2) 0.1% TFA / 80% acetonitrile using an ODP-50 column. After purification by HPLC by gradient elution, the obtained NT-3 fraction was freeze-dried to obtain a freeze-dried powder of NT-3.
[0029]
Example 5
(Character determination of NT-3)
a) Analysis using SDS polyacrylamide gel electrophoresis
NT-3 obtained in Example 4 was sample buffer [Laemmli, Nature,227, 680 (1970)], heated with 100 mM DTT at 100 ° C. for 1 minute, and then electrophoresed with Multigel 10/20 (Daiichi Chemical Co., Ltd.). When the gel after electrophoresis was stained with Coomassie Brilliant Blue, a single band of protein was observed, and the purified product was almost single [FIG. 2]. In FIG. 2, Lanes 1-2 indicate molecular weight markers and NT-3.
[0030]
b) N-terminal amino acid sequence analysis
N-terminal amino acid sequence analysis of NT-3 obtained in Example 4 was performed using a gas phase protein sequencer 477A (Model 477A, Applied Biosystems). As a result, the N-terminal amino acid sequence of NT-3 obtained was consistent with the N-terminal amino acid sequence of NT-3 deduced from the cDNA base sequence. The results are shown in [Table 4].
[Table 4]

[0031]
c) Amino acid composition analysis
About 20 μg of NT-3 obtained in Example 4 was used, and its amino acid composition was determined by an amino acid analyzer (System 6300E, Beckman). As a result, the amino acid composition inferred from the nucleotide sequence of NT-3 cDNA was in good agreement. The results are shown in [Table 5].
[Table 5]

[0032]
d) C-terminal amino acid analysis
Using 15 nmol of NT-3 obtained in Example 4, its C-terminal amino acid was determined by an amino acid analyzer (System 6300E, Beckman). As a result, it was consistent with the C-terminal amino acid deduced from the nucleotide sequence of NT-3 cDNA. The results are shown in [Table 6].
[Table 6]

[0033]
Reference Example 4: Production of Met-human BTC
Met-human BTC was produced according to the method described in Examples 4 to 6, 8 and 13 of JP-A-6-87894.
(1) Construction of E. coli human BTC cDNA expression plasmid
A 0.6 Kb EcoRI-BamHI fragment encoding mature human BTC (1-147 amino acid residues) was isolated from plasmid pTB1515 described in Example 5 of JP-A-6-87894. A synthetic adapter having an ATG translation initiation codon (SEQ ID NO: 5: 5 ′ TATGGATGGG 3 ′; SEQ ID NO: 5: 5 ′ AATTCCCATCA 3 ′) was ligated to the EcoRI site of the above 0.6 Kb fragment, and then generated 0.6 Kb NdeI− The BamHI fragment was ligated with the T7 promoter (Gene,56, 125 (1987)), and plasmid pTB1505 was constructed.
To obtain a DNA fragment encoding 80 amino acid residues of human BTC (from 1 (Asp) to 80 (Tyr) of [Fig. 10-1] to [Fig. 10-2] of JP-A-6-87894) PCR was carried out using plasmid pTB1505 as two primers and two oligonucleotides (SEQ ID NO: 6: 5 ′ ATACATATGGATGGGGAATTCCA 3 ′; SEQ ID NO: 7 ′ 5 ′ CCGGATCCTAGTAAAAACAAGTCAACTCT 3 ′) as primers. The product was digested with NdeI and BamHI and fractionated by 2.0% agarose gel electrophoresis to isolate the desired 0.25 Kb DNA fragment. This 0.25 Kb NdeI-BamHI fragment was ligated and inserted with T4 DNA ligase downstream of the T7 promoter of pET-3c to obtain plasmid pTB1516 (see FIG. 13 of JP-A-6-87894).
[0034]
(2) Expression of human BTC in E. coli
E. coli MM294 was lysogenized with lambda phage (Studier, Supra) recombined with RNA polymerase gene of T7 phage. Thereafter, this plasmid pLysS was introduced into E. coli MM294 (DE3) to obtain E. coli MM294 (DE3) / pLysS. The plasmid pTB1516 obtained in Reference Example 4 (1) was introduced into this microbial cell, whereby E. coli MM294 (DE3) / pLysS, pTB1516 was obtained.
This transformed cell, E. coli MM294 (DE3) / pLysS, pTB1516, was deposited with the microbial industry research institute of MITI on April 21, 1992 under the accession number FERM BP-3836. It has been deposited with the IFO as deposit number IFO 15282 on the 16th of the month.
[0035]
(3) Purification of BTC produced from recombinant E. coli
After E. coli MM294 (DE3) / pLysS, pTB1516 obtained in Reference Example 4 (2) was cultured overnight, the culture solution was inoculated into LB medium so as to be diluted 20-fold. After culturing at 37 ° C. for 2 hours, IPTG was added to a final concentration of 0.1 mM, followed by further culturing for 3 hours. The cells were collected by centrifugation and stored at -20 ° C until use.
The cell stock corresponding to 5-liter culture was thawed and suspended in 300 ml of a buffer containing 50 mM Tris · HCl (pH 7.4), 10 mM EDTA-0.2 M NaCl, 10% sucrose, 1 mM MAPMSF. 40 mg of egg white lysozyme was dissolved in this, incubated at 4 ° C. for 2 hours, then sonicated, and centrifuged at 20,000 × g for 1 hour to obtain a supernatant. The supernatant was passed through a 200 ml Q-Sepharose bed, TCA was added to a final concentration of 4%, and the mixture was allowed to stand at 40 ° C. for 10 minutes. The precipitate collected by centrifuging at 20,000 × g for 20 minutes was suspended in a buffer containing 100 ml of 20 mM Tris (pH 7.4), 1 mM EDTA-0.15 M NaCl, 1 mM APMSF, and homogenized in a mortar. NaOH was added to adjust the pH. The homogenate was centrifuged at 100,000 × g for 1 hour, and the resulting supernatant was applied to an S-Sepharose column (diameter 1.6 × 10 cm, pharmacia). The column was washed with a buffer containing 0.1 M potassium phosphate (pH 6.0), 1 mM EDTA, 0.5 mM PMSF, and then a 0 to 1 M NaCl gradient was applied over 400 ml for 200 minutes, and the eluate was collected every 5 ml. It was. Fraction numbers 20 to 27 with high biological activityE. coli Pooled as BTC fraction.
E. coli TFA was added to the BTC fraction to a final concentration of 0.1% and applied to a C18 reverse phase HPLC column (diameter 1.0 × 25 cm; Asahipak ODP-50, Asahi chemical). After washing the column with 0.1% TFA, a gradient from 0% to 63% acetonitrile was applied over 340 ml for 170 minutes to collect the BTC fraction. 630 μg of this methodE. coli BTC was obtained.
E. coli The N-terminal amino acid sequence of BTC was determined up to 20 amino acid residues. BTC was found to be a molecule with the expected N-terminus starting from the translation initiation methionine.
[0036]
Example 6
After dissolving 20 mg of human betacellulin (Met-BTC) with methionine added at the N-terminus obtained in Reference Example 4 (3) in 16 ml of 50 mM phosphate buffer (pH 8.0), 2 ml of 0.1 M copper sulfate. A mixture of 0.92 g of glyoxylic acid and 2 ml of pyridine was added and reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2 M acetic acid-2 M sodium acetate solution, and the solution used for equilibration was flowed at a flow rate of 6 ml / min. The diketone fractions of Met-BTC were pooled. Subsequently, o-phenylenediamine was added to this fraction so as to have a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the reaction was carried out at 37 ° C. for 20 hours. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM Tris buffer (pH 8.0), and the buffer used for equilibration was flowed at a flow rate of 6 ml / min. Expanded and pooled human betacellulin (BTC) fractions with no methionine added at the N-terminus. The pooled BTC fraction was adjusted to pH 6.0, adsorbed on SP-Sepharose (10 mm ID × 200 mm L) equilibrated with 100 mM phosphate buffer (pH 6.0), and then 0-100% B (B = 100 mM phosphorus) Elution was carried out with a step gradient of acid buffer + 1M NaCl, pH 6.0) for 60 minutes at a flow rate of 2.0 ml / min, and the BTC fractions were pooled. Furthermore, after adsorbing to ODP-50 (10 mm ID × 250 mm L, Showa Denko KK) equilibrated with 0.1% TFA, it has a step gradient of 20-60% B (B = acetonitrile / 0.1% TFA). Elute at a flow rate of 2 ml / min for 40 minutes. The BTC fractions were pooled and then lyophilized to obtain a BTC lyophilized powder.
[0037]
Example 7: (BTC feature determination)
a) Analysis using SDS polyacrylamide gel electrophoresis
1 μg of BTC obtained in Example 6 was added to Sample buffer [Laemmli, Nature,227, 680 (1970)], heated with 100 mM DTT at 100 ° C. for 1 minute, and then electrophoresed with Multigel 15/25 (Daiichi Chemical Co., Ltd.). When the gel after electrophoresis was stained with Coomassie brilliant blue, a single band of protein was observed, and the purified product was almost single. The results are shown in FIG. In FIG. 3, Lanes 1-2 indicate molecular weight markers and BTC.
[0038]
b) N-terminal amino acid sequence analysis
Using 1 nmol of BTC obtained in Example 6, its N-terminal amino acid sequence was determined by a gas phase protein sequencer (Applied Biosystems model 477A). As a result, it was consistent with the N-terminal amino acid sequence deduced from the base sequence of BTC cDNA. The results are shown in [Table 7].
[Table 7]

[0039]
c) Amino acid composition analysis
Using about 20 μg of the BTC obtained in Example 6, the amino acid composition was determined by an amino acid analyzer (Beckman System 6300E). As a result, the amino acid composition was estimated from the base sequence of BTC cDNA. The results are shown in [Table 8].
[Table 8]

[0040]
d) C-terminal amino acid analysis
Using 15 nmol of BTC obtained in Example 6, the C-terminal amino acid was determined by an amino acid analyzer (Beckman System 6300E). As a result, it was consistent with the C-terminal amino acid deduced from the base sequence of the obtained BTC cDNA. The results are shown in [Table 9].
[Table 9]

[0041]
e) BTC biological activity
Molecular cell biology,8588 (1988), BALB / C3T3 A31-714 clone 4 (International Journal of Cancer,12, 463, (1973), and it was confirmed that the purified BTC product obtained in Example 6 had the same activity as that of the standard product.
[0042]
Reference Example 5
Production of methionyl human interleukin-2 (Met-IL-2)
Met-IL-2 was produced according to the method described in Reference Example 2 of JP-A-62-171699.
(I) Construction of expression plasmid
Plasmid pILOT 135-8 carrying human IL-2 gene [see Example 1 (vii) of JP-A-60-115528] is a restriction enzymeH giCut with AI. The resulting 1294 bp DNA fragment was blunted with T4 polymerase, and using T4 DNA ligase,E coThe RI linker dTGCCATGAATTCATGGCA (SEQ ID NO: 8) was conjugated. Obtained DNAE coAfter digestion with RI, a DNA fragment having a translation initiation codon ATG and a human IL-2 gene was obtained.
This DNA fragment isE coRI-P stIt was inserted into ptrp 781 [Nucleic Acids Research, Vol. 11, page 3077 (1983)] digested with the I site using T4 DNA ligase. The plasmid pTF1 for expression thus obtained has a translation disclosure codon and a human IL-2 gene downstream of the trp promoter (see FIG. 4 of JP-A-62-171699).
Plasmid pTF1 is a restriction enzymeS tuCut with I,B amCombined with HI linker. This plasmid DNA is converted into a restriction enzyme.B amHI andE coProcess with RI, thenE coRI-B amThe plasmid was inserted into plasmid pTB281 having a λPL promoter at the HI site. The expression plasmid thus obtained was named pTB285 (see Japanese Patent Application Laid-Open No. 62-171699 [FIG. 5]).
[0043]
(Ii) Production of transformants
Escherichia coli N4830 was obtained from the plasmid pTB285 obtained above by the method of Cohen et al. [Procedures of National Academy of Science (Pro. Natl. Acad. Sci. USA), Vol. 69, p. 2110 (1972)]. The transformant Escherichia coli N4830 / pTB285 containing the above plasmid was obtained.
(Iii) Culture of transformants
Transformant E. coli N4830 / pTB285 (IFO 14437, FERM BP-852) in a 250 ml flask Bacto tryptone (Difco Laboratories, USA) 1%, Bacto yeast extract (Difco Laboratories, USA) 0. The cells were inoculated into 50 ml of a liquid medium (pH 7.0) containing 5%, 0.5% sodium chloride and 50 μg / ml ampicillin and cultured overnight at 37 ° C. with shaking. This culture was transferred to a 5 L jar fermenter containing 2.5 L of M9 medium containing 0.5% casamino acid, 0.5% glucose and 50 μg / ml ampicillin, then at 35 ° C. for 6 hours and then at 42 ° C. for an additional 3 hours. By culture with aeration for a period of time, 2.5 L of a culture solution was obtained. This culture solution was centrifuged, and the cells were collected and stored frozen at -80 ° C.
[0044]
(Iv) Extraction
20 g of frozen cells were uniformly suspended in 100 ml of an extract (pH 7.0) containing 7 M guanidine hydrochloride 0.1 M Tris-HCl, stirred at 4 ° C. for 1 hour, and then centrifuged at 28,000 × g for 20 minutes. A supernatant was obtained.
(V) Partial purification of methionyl interleukin-2 protein
The supernatant obtained was dialyzed against 0.01M Tris-HCl buffer (pH 8.5), and then centrifuged at 19,000 × g for 10 minutes. The supernatant obtained was 0.01M Tris-HCl buffer. After adsorbing the protein through a DE52 (DEAE-cellulose, Whatman, UK) column (50 ml volume) equilibrated with a liquid (pH 8.5), a NaCl linear concentration gradient (0 to 0.15 M NaCl, 1 L) was applied. And Met-IL-2 was eluted to obtain an active fraction.
[0045]
(Vi) Purification of methionyl interleukin-2 protein
The active fraction obtained above was concentrated to 5 ml using YM-5 membrane (Amicon, USA) and equilibrated with 0.1 M Tris-HCl (pH 8.0) -1 M NaCl buffer. Gel filtration was performed using an S-200 (Pharmacia, Sweden) column (500 ml capacity). 40 ml of the active fraction was concentrated to 3 ml with YM-5 membrane. The obtained concentrated liquid was adsorbed on an Ultrapore RPSC (manufactured by Altex, USA) column and subjected to high performance liquid chromatography using a trifluoroacetic acid-acetonitrile system as an elution solvent. Column, Ultrapore RPSC (4.6 × 75 mm); Column temperature, 30 ° C .: Elution solvent A, 0.1% trifluoroacetic acid-99.9% water; Elution solvent B, 0.1% trifluoroacetic acid-99 .9% acetonitrile; elution program, 0 min (68% + 32% B) -25 min (55% A + 45% B) -35 min (45% A + 55% B) -45 min (30% A + 70% B) -48 min (100% B); elution rate, 0.8 ml / min; detection wavelength, 230 nm.
Under these conditions, a Met-IL-2 fraction having a retention time of about 39 minutes was collected.
[0046]
(Vii) Purification of Met-IL-2 by SP-5PW column
High-speed liquid obtained by equilibrating 0.5 ml of 0.005M ammonium acetate buffer (pH 5.0, protein concentration 1.03 mg / ml) containing Met-IL-2 with 0.025 M phosphate buffer (pH 7.4) The protein was eluted using 0.025M phosphate buffer (pH 7.4) on an SP-5PW column for chromatography (0.75 × 7.5 cm; manufactured by Tosoh Corporation). The column temperature was set to 35 ° C., and the buffer flow rate was set to 0.5 ml / min. As the chromatographic system, a Varian 5500 type liquid chromatograph was used.
Under these conditions, the Met-IL-2 fraction having a retention time of about 70 minutes was collected.
[0047]
Example 8
After 20 mg of methionyl human interleukin-2 (Met-IL-2) obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), 1.55 g of glyoxylic acid, 25 mM copper sulfate 3 A mixed solution of .375 ml and 3.375 ml of pyridine was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction so as to have a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the reaction was carried out at 37 ° C. for 21 hours. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h, and human interleukin-2 (IL -2) The fractions were pooled. After adsorbing the pooled IL-2 fractions to SP-5PW equilibrated with 25 mM phosphate buffer (pH 7.0), 30-80% B (B: 25 mM phosphate buffer, pH 8.0) pH gradient For 25 minutes at a flow rate of 1.0 ml / min and the IL-2 fractions were pooled. Further, after adsorption onto an ODP-50 column (4.6 mm ID × 150 mm L) equilibrated with (1) 0.1% TFA and (2) 0.1% TFA + 80% acetonitrile (80%: 20%), 20− Elution was performed with a 100% B concentration gradient for 15 minutes at a flow rate of 0.8 ml / min to obtain purified IL-2. The obtained IL-2 fraction was freeze-dried to obtain a freeze-dried powder of IL-2.
[0048]
Example 9 (Characteristic determination of IL-2)
a) Analysis using SDS polyacrylamide gel electrophoresis
3 μg of IL-2 obtained in Example 8 was added to Sample buffer [Laemmli, Nature,227, 680 (1970)], followed by reduction treatment with 100 mM DTT at 100 ° C. for 5 minutes and electrophoresis with Multigel 10/20 (first chemical). When the gel after electrophoresis was stained with Coomassie Brilliant Blue, it showed the same mobility as that of the standard product and was a single band. The results are shown in FIG. In FIG. 4, Lanes 1-2 indicate IL-2 and molecular weight markers.
[0049]
b) N-terminal amino acid sequence analysis
Using 1 nmol of IL-2 obtained in Example 8, the N-terminal amino acid sequence was determined by a gas phase protein sequencer (Applied Biosystem model 477A). As a result, it was consistent with the N-terminal amino acid sequence predicted from the nucleotide sequence of IL-2 cDNA. The results are shown in [Table 10].
[Table 10]

[0050]
c) Amino acid composition analysis
Using about 20 μg of IL-2 obtained in Example 8, the amino acid composition was determined by an amino acid analyzer (Beckman System 6300E). As a result, it was consistent with the amino acid composition predicted from the cDNA base sequence of IL-2. The results are shown in [Table 11].
[Table 11]

[0051]
d) C-terminal amino acid analysis
Using 15 nmol of IL-2 obtained in Example 8, the C-terminal amino acid was determined by an amino acid analyzer (Beckman System 6300E). As a result, it was consistent with the C-terminal amino acid predicted from the nucleotide sequence of IL-2 cDNA. The results are shown in [Table 12].
[Table 12]

[0052]
e) Biological activity
Biological activity was measured by the method of Hinuma et al. Using bio-independent cells (Biochem. Biophys. Res. Commun.),109, 363 (1982)] and confirmed that the purified product of IL-2 obtained in Example 8 has the same activity as that of the standard product.
[0053]
Reference Example 6 (Construction of human growth hormone (hGH) expression vector using T7 promoter)
The structural gene of hGH was cleaved with EcoRI and EcoRV from plasmid pHGH107 (described in Japanese Patent Publication No. 6-12996, deposit numbers ATCC 31538 and ATCC 40011), and a fragment of about 0.75 kb was isolated. On the other hand, pET-3C [Rosenberg et al. Gene, 56, 125 (1987)] was digested with NdeI and BamHI, and an approximately 4.6 kb fragment having the T7 promoter and ampicillin resistance gene was isolated.
Both fragments were blunt-ended with T4 DNA polymerase (DNA Blunting Kit, manufactured by Takara Shuzo Co., Ltd.), ligated with T4 DNA ligase, introduced into Escherichia coli JM109, and transformants were selected using ampicillin resistance as an index. Twelve of the colonies that emerged were picked up, plasmids were prepared from these, and the cleavage pattern by the restriction enzyme PstI was examined. It was found that the hGH gene was inserted in the correct orientation in the plasmids from the six colonies. . A plasmid obtained from one of these strains was named pTGA201.
[0054]
Reference Example 7 (Expression of Met-hGH in E. coli)
E. coli JM109 was lysogenized with lambda phage (Studier, Supra) recombined with the RNA polymerase gene of T7 phage. Thereafter, the hGH expression vector pTGA201 obtained in Reference Example 6 was introduced into this E. coli JM109 (DE3) to obtain E. coli JM109 (DE3) / pTGA201.
The transformed cells were shaken at 30 ° C. for 16 hours in a 2 liter flask containing 1 liter of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 50 μg / ml ampicillin. Cultured at last. The obtained culture broth was transplanted to a 50-liter fermenter charged with 20 liters of LB medium containing 0.02% Newpol LB-625 (antifoaming agent; manufactured by Sanyo Chemical Industries) and 50 μg / ml ampicillin. The culture was aerated and stirred at 37 ° C. for 6 hours. This culture was added to 360 liters of main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.0246% sulfuric acid. Transplanted to a 500 liter fermentor containing magnesium, 0.02% Newpol LB-625, 0.0005% thiamine hydrochloride, 1.5% glucose, 1.5% casamino acid) and aerated at 37 ° C The culture was started. When the turbidity of the culture reached about 500 kret units, 5.95 mg / liter of isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was further continued. The solution was centrifuged to obtain about 4.5 kg of wet cells and stored frozen at -80 ° C.
The transformed Escherichia coli JM109 (DE3) / pTGA201 has been deposited with the Institute of Industrial Science and Technology (NIBH), the Ministry of International Trade and Industry under the accession number FERM BP-5632, and the Fermentation Research Institute under the accession number IFO 16001. (IFO).
[0055]
Reference Example 8 (Activation of Met-hGH)
6 liters of 50 mM Tris / HCl, 8M guanidine hydrochloride solution (pH 8.0) was added to 2 kg of the microbial cells obtained in Reference Example 7 to dissolve the microbial cells, followed by centrifugation (10,000 rpm, 120 minutes). After adding 18 liters of 50 mM Tris / HCl, 0.28 mM GSSG, 1.4 mM GSH, 0.7 M arginine (pH 8.0) to 6 liters of the obtained supernatant, the pH was adjusted to 8.0 and then active at 4 ° C. for 5 days. Made.
[0056]
Reference Example 9 (Purification of Met-hGH)
Until the electric conductivity becomes 10 mS or less while adding 20 mM Tris / HCl and 2.5 M urea (pH 8.0) to the regenerated solution whose activation has been completed in Reference Example 8 with a Pellicon cassette system (PTGC membrane, Millipore). After desalting and concentration, the obtained concentrated solution was centrifuged (10,000 rpm, 60 minutes) to obtain 5 liters of the supernatant of the concentrated solution. Next, this solution was adsorbed on a DEAE-Toyopearl 650M column (20 cmφ × 84 cm, Tosoh Corp.) equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0), washed thoroughly, and then 0 to 25% B Elution was performed at a flow rate of 300 ml / min for 100 minutes with a concentration gradient (B = 20 mM Tris / HCl, 2.5 M urea, 1 M sodium chloride, pH 8.0), and 10 liters of eluate was obtained as a Met-hGH fraction. Obtained. Further, this eluate was concentrated and desalted with a Pellicon cassette system (PTGC membrane, Millipore). Further, this solution was adsorbed through a DEAE-5PW column (21 cm × 30 cm, Tosoh Corp.) by high performance liquid chromatography (Gilson HPLC system, Gilson), and then A = 50 mM Tris / HCl + 2.5 M urea ( pH 8.0), B = 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) with a 70-85% B pH gradient at a flow rate of 320 ml / min for 70 minutes. Elution was performed to obtain 6 liters of a Met-hGH fraction. To this eluate, 300 ml of 2M Tris / HCl (pH 7.8) solution was added to adjust to pH 7.2, then concentrated and desalted with a Pellicon cassette system (PTGC membrane, Millipore), and 9,979 milligrams of Met-hGH. Got.
[0057]
Example 10 (Removal of N-terminal Met)
To 1650 ml of the Met-hGH solution obtained in Reference Example 9, 413 ml of 2.5M glyoxylic acid, 35 mM copper sulfate and 6M pyridine solution was added and stirred well, and then reacted at 25 ° C. for 60 minutes. Next, the mixture was passed through a Sephadex G-25 column (11.3 cmφ × 125 cm, Pharmacia) equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0) at a flow rate of 3 liter / h for equilibration. The diketone fraction of Met-hGH, which was developed using the same buffer and eluted, was directly added to 4 L of 4 M acetic acid, 4 M sodium acetate, 80 mM Mo-phenylenediamine, and 3 M urea solution with good stirring. After elution was completed, 8 liters of this reaction solution was allowed to stand at 4 ° C. for 3 days. After standing, the mixture was concentrated to 4 liters with a Pellicon cassette system (PTGC membrane, Millipore), and equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0) (11.3 cmφ × 140 cm, Pharmacia) at a flow rate of 3 liters / h, and 4.7 liters of hGH fraction was collected. Further, this solution was adsorbed through a DEAE-5PW column (21 cm × 30 cm, Tosoh Corp.) by high performance liquid chromatography (Gilson HPLC system, Gilson), and then A = 50 mM Tris / HCl + 2.5 M urea ( pH 8.0), B = 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) with a 70-85% B pH gradient at a flow rate of 320 ml / min for 70 minutes. Elution was performed to obtain 10 liters of hGH fraction. To this hGH fraction, 500 ml of a 2M Tris / HCl (pH 7.8) solution was added to adjust to pH 7.2, and then concentrated with Minitan II (PTGC membrane, Millipore), and 500 ml of this concentrated solution was equilibrated with distilled water. The solution was passed through a Sephacryl S-100 column (113.cmφ × 50cm, Pharmacia) at a flow rate of 2 liter / h and developed to obtain 1651 ml of hGH fraction. Further, this solution was filtered through Millipak 60 (Millipore) to obtain 1487 ml of hGH solution (3309 mg of hGH).
[0058]
Example 11 (Characteristic determination of hGH)
(A) Analysis using SDS polyacrylamide gel electrophoresis
Sample buffer containing 100 mM DTT in hGH obtained in Example 10 [Laemmli, Nature,227, 680 (1970)] was added and stirred well, heated at 95 ° C. for 2 minutes, and then electrophoresed with Multigel 10/20 (first chemical). As a result of staining the gel after electrophoresis with Coomassie brilliant blue, a single band was observed at about 22 kd, confirming that the purified hGH was almost single (FIG. 5). . In FIG. 5, lanes 1-2 show molecular weight markers and hGH.
[0059]
(B) Amino acid composition analysis
The amino acid composition was determined using an amino acid analyzer (L-8500A, Hitachi). As a result, the amino acid composition of the obtained hGH coincided with the amino acid composition estimated from the base sequence of cDNA (Table 13).
[Table 13]

[0060]
(C) N-terminal amino acid sequence analysis
The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystems, model 477A). As a result, the N-terminal amino acid sequence of the obtained hGH coincided with the N-terminal amino acid sequence of hGH deduced from the cDNA base sequence (Table 14).
[Table 14]

[0061]
(D) C-terminal amino acid analysis
The C-terminal amino acid was determined using an amino acid analyzer (L-8500A, Hitachi). The C-terminal amino acid of the obtained hGH coincided with the C-terminal amino acid deduced from the cDNA base sequence (Table 15).
[Table 15]

[0062]
Example 12 (Measurement of hGH activity)
The growth promoting effect of purified hGH obtained in Example 10 on Nb2 cells [Journal of Clinical Endocrinology and Metabolism, Vol. 51, pp. 1058 (1980)] was measured according to a standard product (Chemicon International, Temecula). , California, USA).
[0063]
Example 13 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. 1 ml of 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM nickel chloride, and 6 M urea (pH 7.0) solution was added to 1 ml of this solution and stirred well, followed by reaction at room temperature for 60 minutes. Subsequently, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.0), and the eluted diketone of Met-hGH. Fractions were collected, and equal amounts of 4M acetic acid, 4M sodium acetate, and 80mMo-phenylenediamine solution were added to the eluate and stirred well, followed by reaction at 37 ° C for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0064]
Example 14 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. 1 ml of 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM cobalt chloride, 6 M urea (pH 7.0) solution was added to 1 ml of this solution, and the mixture was stirred well and then allowed to react at room temperature for 60 minutes. Subsequently, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.0), and the eluted diketone of Met-hGH. Fractions were collected, and equal amounts of 4M acetic acid, 4M sodium acetate, and 80mMo-phenylenediamine solution were added to the eluate and stirred well, followed by reaction at 37 ° C for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0065]
Example 15 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. 1 ml of 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM zinc sulfate, and 6 M urea (pH 7.0) solution was added to 1 ml of this solution and stirred well, followed by reaction at room temperature for 60 minutes. Subsequently, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.0), and the eluted diketone of Met-hGH. Fractions were collected, and equal amounts of 4M acetic acid, 4M sodium acetate, and 80mMo-phenylenediamine solution were added to the eluate and stirred well, followed by reaction at 37 ° C for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0066]
Example 16 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. To 1 ml of this solution, 1 ml of 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM copper acetate, 6M urea (pH 7.0) solution was added and stirred well, followed by reaction at room temperature for 60 minutes. Subsequently, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.0), and the eluted diketone of Met-hGH. Fractions were collected, and equal amounts of 4M acetic acid, 4M sodium acetate, and 80mMo-phenylenediamine solution were added to the eluate and stirred well, followed by reaction at 37 ° C for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0067]
Example 17 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. To 1 ml of this solution, 1 ml of 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM copper sulfate, 6M urea (pH 7.0) solution was added and stirred well, followed by reaction at room temperature for 60 minutes. Next, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.0), and the eluted diketone of Met-hGH. Fractions were collected, and an equal amount of 4M acetic acid, 4M sodium acetate, 80mM tolylene-3,4-diamine solution was added to the eluate and stirred well, followed by reaction at 37 ° C for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0068]
Example 18 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. To 1 ml of this solution, 1 ml of 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM copper sulfate, 6M urea (pH 7.0) solution was added and stirred well, followed by reaction at room temperature for 60 minutes. Subsequently, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.0), and the eluted diketone of Met-hGH. Fractions were collected, and an equal amount of 4M acetic acid, 4M sodium acetate, and 80 mM 4-chloro-ophenylenediamine solution were added to the eluate and stirred well, followed by reaction at 37 ° C. for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0069]
Example 19 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. To 1 ml of this solution, 1 ml of 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM copper sulfate, 6M urea (pH 7.0) solution was added and stirred well, followed by reaction at room temperature for 60 minutes. Subsequently, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.0), and the eluted diketone of Met-hGH. Fractions were collected, and an equal amount of 4M acetic acid, 4M sodium acetate, 80 mM 3,4-diaminobenzoic acid solution was added to the eluate, and the mixture was stirred well and reacted at 37 ° C for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0070]
Example 20 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 8M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- It concentrated so that the density | concentration of hGH might be 10 mg / ml. To 1 ml of this solution, 1 ml of 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM copper sulfate, 6M urea (pH 7.0) solution was added and stirred well, followed by reaction at room temperature for 60 minutes. Next, this reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 4M urea (pH 8.5), and the eluted diketone of Met-hGH. Fractions were collected, cysteamine was added to the eluate to a concentration of 40 mM, and the mixture was stirred well and reacted at 37 ° C. for 15 hours. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl (pH 8.0), and the hGH fraction was collected, followed by further high performance liquid chromatography ( This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) using a Gilson HPLC system (Gilson), then A = 50 mM Tris / HCl + 2.5 M urea (pH 8.0), B = Elution with a pH gradient of 70-85% B with 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) for 70 minutes at a flow rate of 7.5 ml / min. hGH was obtained.
[0071]
Example 21 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 3M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- The hGH concentration was concentrated to 5 mg / ml. 2 ml of 4M sodium acetate, 0.8M acetic acid, 0.4M glyoxylic acid, 10 mM copper sulfate and 2.5M urea solution was added to 2 ml of this solution and stirred well, followed by reaction at room temperature for 60 minutes. Next, the reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0), and the eluted Met-hGH was eluted. Diketone fractions were collected, and an equal amount of 4M acetic acid, 4M sodium acetate, 3M urea, and 80mMo-phenylenediamine solution were added to the eluate and stirred well, followed by reaction at 4 ° C for 3 days. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0). This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) by liquid chromatography (Gilson HPLC system, Gilson), and then A = 50 mM Tris / HCl + 2.5 M urea (pH 8). 0.0), B = 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) with a 70-85% B pH gradient for 70 minutes, 7.5 ml / min flow rate And elution was performed to obtain hGH.
[0072]
Example 22 (Removal of N-terminal Met)
The Met-hGH solution obtained by the same method as in Reference Example 9 was replaced with 20 mM Tris / HCl, 3M urea (pH 8.0) using an ultrafiltration system (Diaflow Membrane YM10, 43 mm, Amicon) and Met- The hGH concentration was concentrated to 5 mg / ml. 2 ml of 1 M imidazole, 0.5 M glyoxylic acid, 20 mM copper sulfate, and 2.5 M urea solution was added to 2 ml of this solution and stirred well, followed by reaction at room temperature for 60 minutes. Next, the reaction solution was passed through a Sephadex G-25 column (10 mm ID × 30 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0), and the eluted Met-hGH was eluted. Diketone fractions were collected, and an equal amount of 4M acetic acid, 4M sodium acetate, 3M urea, and 80mMo-phenylenediamine solution were added to the eluate and stirred well, followed by reaction at 4 ° C for 3 days. After the reaction, the solution was passed through a Sephadex G-25 column (10 mm ID × 40 cmL, Pharmacia) equilibrated with 20 mM Tris / HCl, 2.5 M urea (pH 8.0). This solution was adsorbed through a DEAE-5PW column (2.15 cm × 15 cm, Tosoh Corp.) by liquid chromatography (Gilson HPLC system, Gilson), and then A = 50 mM Tris / HCl + 2.5 M urea (pH 8). 0.0), B = 50 mM MES [2- (N-morpholino) ethanesulfonic acid] +2.5 M urea (pH 4.0) with a 70-85% B pH gradient for 70 minutes, 7.5 ml / min flow rate And elution was performed to obtain hGH.
[0073]
Example 23 (Removal of N-terminal Met)
10 mg of Met-BTC added with Met at the N-terminus obtained in Reference Example 4 was dissolved in 4 ml of 3M urea solution, and then a mixed solution of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid and 0.5 ml of pyridine (pH 5. 0) was added and reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration. Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, after adding an equal amount of 4M acetic acid-4M sodium acetate solution to this fraction, o-phenylenediamine was added so as to have a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the reaction was performed at 37 ° C for 15 hours. Reacted. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 50 mM phosphate buffer (pH 6.0), and the buffer used for equilibration was flowed at a flow rate of 6 ml / min. The BTC fractions without Met added to the N-terminus were pooled. After the pooled BTC fractions were adjusted to pH 6.0 and adsorbed to SP-5PW (7.5 mm ID × 75 mm L, Tosoh Corp.) equilibrated with 100 mM phosphate buffer + 200 mM NaCl (pH 5.0), Elution was performed at a flow rate of 0.8 ml / min for 30 minutes with a step gradient of 0 to 100% B (B = 100 mM phosphate buffer + 200 mM NaCl, pH 9.0), and the BTC fractions were pooled. After adsorbing to ODP-50 (10 mm ID × 250 mm L) equilibrated with 0.1% TFA, Showa Denko KK), 20-60% B (B = 80% acetonitrile / 0.1% TFA) Elute with a step gradient for 40 minutes at a flow rate of 2 ml / min. The BTC fractions were pooled and then lyophilized to obtain about 760 μg of BTC.
[0074]
Example 24 (BTC feature determination)
(A) Analysis using SDS polyacrylamide gel electrophoresis
Sample buffer added with 100 mM DTT was added to the BTC obtained in Example 23 [Laemmli, Nature,227, 680 (1970)], heated at 95 ° C. for 1 minute, and then electrophoresed with Multigel 15/25 (first chemical). As a result of staining the gel after electrophoresis with Coomassie brilliant blue, a single band protein was observed at about 16 Kd. From this, it was found that purified BTC was almost single and formed a strong dimer even under reducing conditions with DTT (FIG. 6). In FIG. 6, lanes 1-2 show molecular weight markers and BTC.
[0075]
(B) Amino acid composition analysis
The amino acid composition was determined using an amino acid analyzer (Beckman System 6300E). As a result, the amino acid composition was inferred from the base sequence of BTC cDNA (Table 16).
[Table 16]

[0076]
(C) N-terminal amino acid sequence analysis
The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystem model 477A). As a result, it was consistent with the N-terminal amino acid sequence of BTC deduced from the base sequence of BTC cDNA (Table 17).
[Table 17]

[0077]
(D) C-terminal amino acid analysis
The C-terminal amino acid was determined using an amino acid analyzer (Beckman System 6300E). The C-terminal amino acid of the obtained BTC coincided with the C-terminal amino acid deduced from the cDNA base sequence (Table 18).
[Table 18]

[0078]
Example 25 (Bioactivity of BTC)
BALB / c 3T3 A31-714 clone 4 (International Journal of Cancer, 12, 463 (1973)) by the method described in Molecular Cell Biology, 8, 588 (1988)) The purified BTC obtained in Example 23 was confirmed to have cell growth promoting activity equivalent to that of a standard product (purified BTCI described in Example 13 of JP-A-6-87894).
[0079]
Example 26
40 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid and 0.5 ml of pyridine was added. , Reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, and then tolylene-3,4-diamine was added to a concentration of 40 mM, followed by deaeration and nitrogen gas sealing at 37 ° C. For 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0080]
Example 27
40 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid and 0.5 ml of pyridine was added. , Reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, after adding an equal amount of 4M acetic acid-4M sodium acetate solution to this fraction, 2,3-diaminophenol was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were carried out at 37 ° C. Reacted for 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0081]
Example 28
40 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid and 0.5 ml of pyridine was added. , Reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, after adding an equal amount of 4M acetic acid-4M sodium acetate solution to this fraction, 3,4-diaminobenzoic acid was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were carried out at 37 ° C. For 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0082]
Example 29
40 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid and 0.5 ml of pyridine was added. , Reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, after adding an equal amount of 4M acetic acid-4M sodium acetate solution to this fraction, 4-chloro-o-phenylenediamine was added to a concentration of 40 mM, and deaeration and nitrogen gas sealing were performed. The reaction was carried out at 0 ° C. for 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0083]
Example 30
40 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid and 0.5 ml of pyridine was added. , Reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, cysteamine was added to this fraction to a concentration of 40 mM, adjusted to pH 8.5, degassed and sealed with nitrogen gas, and reacted at 37 ° C. for 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0084]
Example 31
20 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 2 ml of 3M urea solution, and then 2 ml of a solution of 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM nickel sulfate, 6M urea. And reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0085]
Example 32
20 mg of Met-BTC with methionine added to N-terminus obtained in Reference Example 4 was dissolved in 2 ml of 3M urea solution, and then 2 ml of 4M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM cobalt chloride, 6M urea solution. And reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0086]
Example 33
20 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 2 ml of 3M urea solution, and then 2 ml of a solution of 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM zinc sulfate, 6M urea. And reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0087]
Example 34
40 mg of Met-BTC3 with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper acetate, 0.25 g of glyoxylic acid and 0.5 ml of pyridine was added. , Reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0088]
Example 35
After dissolving 20 mg of Met-BTC with methionine added to the N-terminal obtained in Reference Example 4 in 2 ml of 3M urea solution, 4M sodium acetate, 0.8M acetic acid, 0.4M glyoxylic acid, 10 mM copper sulfate, 3M urea 2 ml of the solution was added and reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0089]
Example 36
20 mg of Met-BTC with methionine added to the N-terminus obtained in Reference Example 4 was dissolved in 2 ml of 3M urea solution, and then 2 ml of a solution of 1M imidazole, 0.5M glyoxylic acid, 20 mM copper sulfate, 3M urea was added. It reacted at 25 degreeC for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-BTC were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 23 to obtain BTC.
[0090]
Example 37
40 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine. And reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 50 mM phosphate buffer (pH 6.0), and the buffer used for equilibration was flowed at a flow rate of 6 ml / min. The NT-3 fractions with no methionine added at the N-terminus were pooled. After adjusting the pooled NT-3 fraction to pH 6.0 and adsorbing to SP-5PW (21.5 mmID × 150 mmL, Tosoh Corp.) equilibrated with 100 mM phosphate buffer + 200 mM NaCl (pH 5.0) The elution was performed at a flow rate of 6 ml / min for 55 minutes with a step gradient of 0-100% B (B = 100 mM phosphate buffer + 200 mM NaCl, pH 9.0), and the NT-3 fractions were pooled. Furthermore, after adsorbing the NT-3 fraction to ODP-50 (10 mm ID × 250 mm L, Showa Denko KK) equilibrated with 0.1% TFA, 20-60% B (B = 80% acetonitrile / 0.00%). Elute at a flow rate of 2 ml / min for 40 minutes with a step gradient of 1% TFA). NT-3 fractions were pooled and then lyophilized to obtain about 600 μg of NT-3.
[0091]
Example 38 (Characteristic determination of NT-3)
a) Analysis using SDS polyacrylamide gel electrophoresis
Sample buffer [Laemmli, Nature, containing 100 mM DTT and NT-3 obtained in Example 37]227, 680 (1970)] and heated at 100 ° C. for 1 minute, followed by electrophoresis with Multigel 15/25 (first chemical). When the gel after electrophoresis was stained with Coomassie brilliant blue, a single band of protein was observed, and the purified product was almost single (FIG. 7). In FIG. 7, lanes 1-2 show molecular weight markers and NT-3.
[0092]
N-terminal amino acid sequence analysis
The N-terminal amino acid sequence of NT-3 obtained in Example 37 was determined using a gas phase protein sequencer (Applied Biosystems model 477A). As a result, the N-terminal amino acid sequence of NT-3 obtained was consistent with the N-terminal amino acid sequence of NT-3 deduced from the base sequence of cDNA. (Table 19)
[Table 19]

[0093]
b) Amino acid composition analysis
The amino acid composition of NT-3 obtained in Example 37 was determined using an amino acid analyzer (Beckman System 6300E). As a result, the amino acid composition was estimated from the nucleotide sequence of NT-3 cDNA (Table 20).
[Table 20]

[0094]
C-terminal amino acid analysis
The C-terminal amino acid of NT-3 obtained in Example 37 was determined using an amino acid analyzer (Beckman System 6300E). The C-terminal amino acid sequence of the obtained NT-3 was consistent with the C-terminal amino acid deduced from the cDNA base sequence (Table 21).
[Table 21]

[0095]
Example 39 (Measurement of biological activity of NT-3)
About NT-3 obtained in Example 37, the biological activity measurement using DRG was performed, and it confirmed that it had activity equivalent to NT-3 obtained from the CHO cell.
[0096]
Example 40
40 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine. And reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, and then tolylene-3,4-diamine was added to a concentration of 40 mM, followed by deaeration and nitrogen gas sealing at 37 ° C. For 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0097]
Example 41
40 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine. And reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, after adding an equal amount of 4M acetic acid-4M sodium acetate solution to this fraction, 2,3-diaminophenol was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were carried out at 37 ° C. Reacted for 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0098]
Example 42
40 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine. And reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, after adding an equal amount of 4M acetic acid-4M sodium acetate solution to this fraction, 3,4-diaminobenzoic acid was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were carried out at 37 ° C. For 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0099]
Example 43
40 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine. And reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, after adding an equal amount of 4M acetic acid-4M sodium acetate solution to this fraction, 4-chloro-o-phenylenediamine was added to a concentration of 40 mM, and deaeration and nitrogen gas sealing were performed. The reaction was carried out at 0 ° C. for 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0100]
Example 44
40 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 4 ml of 3M urea solution, and then a mixture of 0.5 ml of 50 mM copper sulfate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine. And reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, cysteamine was added to this fraction to a concentration of 40 mM and the pH was adjusted to 8.5, followed by degassing and nitrogen gas sealing, and the reaction was carried out at 37 ° C. for 15 hours. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0101]
Example 45
20 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 2 ml of 3 M urea solution, and then 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM nickel sulfate, and 6 M urea were added. 2 ml of the solution was added and reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0102]
Example 46
After 20 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 2 ml of 3M urea solution, 4M sodium acetate, 20mM acetic acid, 0.4M glyoxylic acid, 20mM cobalt chloride, 6M urea 2 ml of the solution was added and reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0103]
Example 47
20 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 2 ml of 3 M urea solution, and then 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM zinc sulfate, and 6 M urea were added. 2 ml of the solution was added and reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0104]
Example 48
40 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 4 ml of 3M urea solution, and then a mixed solution of 0.5 ml of 50 mM copper acetate, 0.25 g of glyoxylic acid, and 0.5 ml of pyridine. And reacted at 25 ° C. for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0105]
Example 49
After dissolving 20 mg of Met-NT-3 with methionine added to the N-terminal obtained in Reference Example 3 in 2 ml of 3M urea solution, 4M sodium acetate, 0.8M acetic acid, 0.4M glyoxylic acid, 10 mM copper sulfate, 3M 2 ml of urea solution was added and reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0106]
Example 50
20 mg of Met-NT-3 with methionine added to the N-terminus obtained in Reference Example 3 was dissolved in 2 ml of 3M urea solution, and then 2 ml of a solution of 1M imidazole, 0.5M glyoxylic acid, 20 mM copper sulfate and 3M urea was added. And reacted at 25 ° C. for 1 hour.
After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 2.5 M urea + 50 mM phosphate buffer (pH 6.0), and the solution used for equilibration Was developed at a flow rate of 6 ml / min, and the diketone fractions of Met-NT-3 were pooled. Subsequently, an equal amount of 4M acetic acid-4M sodium acetate solution was added to this fraction, o-phenylenediamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was subjected to 37 ° C. for 15 hours. Reacted. After completion of the reaction, purification was performed in the same manner as in Example 37 to obtain NT-3.
[0107]
Activation of the modified Met-NT-3 and purification of the activated Met-NT-3 can also be performed, for example, by the following method. That is, after centrifuging (10000 rpm) the pellet solution obtained in Reference Example 3 (5), the supernatant liquid was 1.8 M urea, 0.2 M Arg, 0.2 mM GSSG, 1.0 mM GSH, 100 mM phosphoric acid. It is diluted about 20 times with a buffer solution (pH 8.5), and refolding of denatured Met-NT-3 can be performed at 4 ° C. for 4 weeks. The refolding solution after completion of refolding was adjusted to pH 6.0, adsorbed on an SP-Sepharose column (22 mm ID × 120 mm L) equilibrated with 100 mM phosphate buffer (pH 6.0), and then 400 mM NaCl / 100 mM phosphorus. ODP-50 (21.5 mm ID × 300 mm L) eluted with an acid buffer (pH 6.0), pooled with fractions containing Met-NT-3, and then equilibrated with 0.1% TFA (Showa Denko K.K. )) And lyophilize the NT-3 fraction obtained by elution with a step gradient of 0-80% B (B = acetonitrile / 0.1% TFA) for 60 minutes at a flow rate of 5 ml / min. , Met-NT-3 powder can be obtained. Furthermore, refolding can be performed using citrulline, alanine, valine, lysine, aspartic acid or the like instead of the above-mentioned arginine.
[0108]
Example 51
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 .1M nickel chloride (3.375 ml), 4M sodium acetate, 20 mM acetic acid, 6M urea solution (3.375 ml) was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0109]
Example 52
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 .1M Cobalt Chloride 3.375ml, 4M Sodium Acetate, 20mM Acetic Acid, 6M Urea Solution, 3.375ml was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0110]
Example 53
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 .1M zinc sulfate (3.375 ml), 4M sodium acetate, 20 mM acetic acid, 6M urea solution (3.375 ml) was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0111]
Example 54
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 .1M copper acetate (3.375 ml), 4M sodium acetate, 20 mM acetic acid, 6M urea solution (3.375 ml) was added, and the mixture was reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0112]
Example 55
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 . A mixed solution of 3.3M ml of 1M copper sulfate and 3.375 ml of pyridine was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, this fraction was adjusted to pH 8.5, cysteamine was added to a concentration of 40 mM, deaeration and nitrogen gas sealing were performed, and the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0113]
Example 56
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 . A mixed solution of 3.3M ml of 1M copper sulfate and 3.375 ml of pyridine was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, to this fraction, tolylene-3,4-diamine was added to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, the reaction was carried out at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0114]
Example 57
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 . A mixed solution of 3.3M ml of 1M copper sulfate and 3.375 ml of pyridine was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, 4-chloro-o-phenylenediamine was added to this fraction to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, the reaction was carried out at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0115]
Example 58
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 . A mixed solution of 3.3M ml of 1M copper sulfate and 3.375 ml of pyridine was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, 3,4-diaminobenzoic acid was added to this fraction to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, reaction was performed at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0116]
Example 59
20 mg of human interleukin 2 (Met-IL-2) with methionine added to the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then glyoxylic acid 1.55 g, 0 . A mixed solution of 3.3M ml of 1M copper sulfate and 3.375 ml of pyridine was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, 2,3-diaminophenol was added to this fraction to a concentration of 40 mM, and after deaeration and nitrogen gas sealing, reaction was performed at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0117]
Example 60
20 mg of human interleukin 2 (Met-IL-2) added with methionine at the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then 0.5 M glyoxylic acid, 20 mM. An equal amount of zinc sulfate, 40 mM ammonium acetate buffer (pH 5), and 6M urea solution was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0118]
Example 61
20 mg of human interleukin 2 (Met-IL-2) added with methionine at the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then 0.5 M glyoxylic acid, 20 mM. Copper acetate, 40 mM ammonium acetate buffer (pH 5), and 6 M urea solution were added in equal amounts and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0119]
Example 62
20 mg of human interleukin 2 (Met-IL-2) added with methionine at the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then 0.5 M glyoxylic acid, 20 mM. An equal amount of nickel chloride, 40 mM ammonium acetate buffer (pH 5), and 6M urea solution was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0120]
Example 63
20 mg of human interleukin 2 (Met-IL-2) added with methionine at the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then 0.5 M glyoxylic acid, 20 mM. Cobalt chloride, 40 mM ammonium acetate buffer solution (pH 5), and 6 M urea solution were added in equal amounts and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0121]
Example 64
20 mg of human interleukin 2 (Met-IL-2) added with methionine at the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then 20 mM Tris HCl containing 3 M urea. (PH 8) Dialyzed against 2 L. Equal amounts of 1M imidazole, 0.5M glyoxylic acid, 20 mM copper sulfate, and 2.5M urea solution were mixed in this dialysis internal solution and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0122]
Example 65
20 mg of human interleukin 2 (Met-IL-2) added with methionine at the N-terminus obtained in Reference Example 5 was dissolved in 27 ml of 20 mM ammonium acetate buffer (pH 5.0), and then 20 mM Tris HCl containing 3 M urea. (PH 8) Dialyzed against 2 L. 4M sodium acetate, 0.4M glyoxylic acid, 10mM copper sulfate, 0.8M acetic acid, and 2.5M urea solution were mixed in equal amounts in this dialysis internal solution and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was passed through a Sephadex G-25 column (25 mm ID × 600 mm L) equilibrated with 20 mM ammonium acetate buffer (pH 5.0) at a flow rate of 300 ml / h to obtain a diketone of Met-IL-2. Body fractions were pooled. Subsequently, o-phenylenediamine was added to this fraction to a concentration of 20 mM, and after deaeration and nitrogen gas sealing, the mixture was reacted at 37 ° C. for 21 hours. After completion of the reaction, a freeze-dried IL-2 powder was obtained according to the method of Example 8.
[0123]
Reference Example 10
Escherichia coli MM294 (DE3) / pE-C35PTH (IFO 15213; FERM BP-3508) (JP-A-5-304976) was mixed with 1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, 50 μg / ml. 1 L of a liquid medium (pH 7) containing ampicillin was inoculated and cultured at 30 ° C. with rotary shaking. 1 L of this culture solution was inoculated into 19 L of a liquid medium (pH 7) containing 1% bactotryptone, 0.5% yeast extract and 0.5% sodium chloride, and cultured with aeration and stirring at 30 ° C. for 8 hours. This culture solution was added to M-9 medium (1.5% casamino acid, 1.5% glucose, 0.0005% vitamin B).₁) 17 L was transferred to a 500 L fermentor containing 343 L, and aerated and stirred for 7 hours at 37 ° C. to obtain about 350 L of a culture solution. This culture solution was centrifuged to obtain about 4 kg of wet cells and stored frozen at -80 ° C.
The above Escherichia coli MM294 (DE3) / pE-C35PTH has accession number IFO
15213 is deposited with the Fermentation Research Institute (IFO).
[0124]
Reference Example 11
Add 40 ml of 20 mM ammonium acetate buffer (pH 5) containing 7M guanidine hydrochloride to 10 g of frozen cells, dissolve the cells, and centrifuge (10,000 rpm, 60 minutes) to obtain about 40 ml of cell extract. Obtained. This bacterial cell extract was diluted with about 2 L of 20 mM ammonium acetate buffer (pH 5), and then centrifuged (4,200 rpm, 20 minutes) to obtain about 2 L of a centrifugal supernatant. The supernatant was passed through an SP-Toyopearl 650M column (5 cm × 30 cm), adsorbed and washed, and then eluted with 20 mM ammonium acetate buffer (pH 5) containing 1 M sodium chloride. This eluate was subjected to concentration gradient elution using an ODS-120T column (21.5 cm × 30 cm, Tosoh) and (1) 0.1% trifluoroacetic acid and (2) 80% acetonitrile containing 0.1% trifluoroacetic acid. Adsorbed and eluted by high-performance liquid chromatography using a solution of about 30 ml of Met [Cys³⁵A PTH (1-84) fraction was obtained. This solution was dialyzed against 0.1 M acetic acid containing 5 L of 6 M urea to obtain a dialysate. About 4 mg of DMAP-CN (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was added to the resulting dialysate, reacted at room temperature for 15 minutes, and Met- [Cys (CN)³⁵] PTH (1-84) was obtained. The obtained reaction solution was passed through SP-Toyopearl 650M (2.0 cm × 30 cm), adsorbed, washed with 20 mM ammonium acetate buffer (pH 5) containing 100 mM sodium chloride, and the reagent was removed. Elution was performed with 20 mM ammonium acetate buffer (pH 5) containing 1 M sodium chloride. This eluate was dialyzed against 5 L of 6M urea to obtain about 8 ml of dialysate. The obtained dialysate was ice-cooled, 400 μl of 1N sodium hydroxide was added, and the mixture was reacted at 0 ° C. for 10 minutes. The obtained reaction solution was subjected to gel filtration using Toyopearl HW-50F (2 cm × 50 cm) equilibrated with 20 mM ammonium acetate buffer (pH 5), and MetPTH (1-34) fractions were pooled. The obtained MetPTH (1-34) fraction was added to an ODS-120T column (21.5 cm × 30 cm, Tosoh) and (1) 80 containing 0.1% trifluoroacetic acid and (2) 0.1% trifluoroacetic acid. Adsorption and elution were carried out by high performance liquid chromatography using a concentration gradient elution method with% acetonitrile to obtain about 50 ml of a MetPTH (1-34) fraction. This eluate was freeze-dried to obtain about 5 mg of MetPTH (1-34).
[0125]
Example 66
5 mg of Met-PTH (1-34) with methionine added to the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of distilled water, and then mixed with 46.4 mg of glyoxylic acid, 2.5 mg of copper sulfate and 94.8 mg of pyridine. The solution was added and reacted at room temperature for 1 hour. After completion of the reaction, the mixture was centrifuged, and the supernatant was ODP-50 column (1 cm × 25 cm, Showa Denko) and (1) 20 mM ammonium acetate (pH 5) containing 10% acetonitrile and (2) 20 mM containing 60% acetonitrile. It was adsorbed and eluted by high performance liquid chromatography using a concentration gradient elution method with ammonium acetate (pH 5), and about 5.4 ml of diketone fractions of Met-PTH (1-34) were pooled. Next, this fraction was freeze-dried, and distilled water was added to the resulting freeze-dried product to make 4 ml. Then, 4 ml of a mixture of 4M acetic acid and 4M sodium acetate containing 80 mM o-phenylenediamine was added to remove the product. After performing nitrogen gas sealing, the reaction was carried out at 37 ° C. for 17 hours. After completion of the reaction, the concentration of the reaction solution with an ODP-50 column (1 cm × 25 cm, Showa Denko) and (1) 0.1% trifluoroacetic acid and (2) 80% acetonitrile containing 0.1% trifluoroacetic acid Adsorption and elution were performed by high performance liquid chromatography using an elution method, and PTH (1-34) fractions were pooled. The pooled PTH (1-34) fraction was diluted 5-fold with 20 mM ammonium acetate (pH 5) and equilibrated with 20 mM ammonium acetate buffer (pH 5) containing 2 M urea (7.5 mm × 75 mm, 7.5 mm × 75 mm, After adsorbing to Tosoh), PTH (1-34) was carried out at a flow rate of 0.8 ml / min for 30 minutes with a concentration gradient of 0-50% B (B: 20 mM MES-2M urea-0.5 M sodium chloride, pH 8). Fractions were pooled. This fraction was dialyzed against 1 L of distilled water and freeze-dried to obtain a freeze-dried powder of PTH (1-34).
[0126]
Example 67 (Characteristic determination of PTH (1-34))
a) Analysis using SDS polyacrylamide gel electrophoresis
PTH (1-34) obtained in Example 66 was suspended in a sample buffer [NOVEX JAPAN], and then electrophoresed with Peptide-PAGE mini [TEFCO]. When the gel after electrophoresis was stained with Coomassie Brilliant Blue, it was a single band (FIG. 8). In FIG. 8, lanes 1-2 show molecular weight markers and PTH (1-34).
[0127]
b) N-terminal amino acid sequence analysis
The N-terminal amino acid sequence was determined using a gas phase protein sequencer (Applied Biosystem model 477A). As a result, it was consistent with the N-terminal amino acid sequence predicted from the base sequence of PTH (1-34). (Table 22)
[Table 22]

[0128]
c) Amino acid composition analysis
The amino acid composition was determined using an amino acid analyzer (Hitachi L-8500A Amino Acid Analyzer). As a result, it was consistent with the amino acid composition predicted from the cDNA base sequence of PTH (1-34) (Table 23).
[Table 23]

[0129]
d) C-terminal amino acid analysis
The C-terminal amino acid was determined using an amino acid analyzer (Hitachi L-8500A Amino Acid Analyzer). As a result, it was consistent with the C-terminal amino acid predicted from the base sequence of PTH (1-34). (Table 24)
[Table 24]

Biological activity is measured by the method using osteoblast-like cell line MC3T3-E1 cells [Nakagawa, S et al., BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONDS,200, 1735-1741 (1994)] and was confirmed to have the same activity as the standard.
[0130]
Example 68
5 mg of Met-PTH (1-34) with methionine added to N-terminal obtained in Reference Example 11 was dissolved in 1 ml of 4M urea, 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM nickel chloride, 4M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM o-phenylenediamine was added, and after degassing nitrogen gas sealing, 37 The reaction was carried out at ° C for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0131]
Example 69
5 mg of Met-PTH (1-34) with methionine added at the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4 M urea, and 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM cobalt chloride, 4 M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM o-phenylenediamine was added, and after degassing nitrogen gas sealing, 37 The reaction was carried out at ° C for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0132]
Example 70
5 mg of Met-PTH (1-34) with methionine added at the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4M urea, 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM zinc sulfate, 4M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM o-phenylenediamine was added, and after degassing nitrogen gas sealing, 37 The reaction was carried out at ° C for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0133]
Example 71
5 mg of Met-PTH (1-34) with methionine added to N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4M urea, and 4M sodium acetate, 20 mM acetic acid, 0.4M glyoxylic acid, 20 mM copper acetate, 4M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM o-phenylenediamine was added, and after degassing nitrogen gas sealing, 37 The reaction was carried out at ° C for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0134]
Example 72
5 mg of Met-PTH (1-34) with methionine added to the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4 M urea, and 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM copper sulfate, 4 M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of 50 mM Tris.HCl (pH 8.5), cysteamine was added to a final concentration of 40 mM, deaerated nitrogen gas sealing was performed, and then the mixture was sealed at 37 ° C. Reacted for hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0135]
Example 73
5 mg of Met-PTH (1-34) with methionine added to the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4 M urea, and 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM copper sulfate, 4 M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of a solution of 4M acetic acid and 4M sodium acetate containing 80 mM tolylene-3,4-diamine was added, and degassing nitrogen gas sealing was performed. Then, it reacted at 37 degreeC for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0136]
Example 74
5 mg of Met-PTH (1-34) with methionine added to the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4 M urea, and 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM copper sulfate, 4 M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM 4-chloro-o-phenylenediamine was added, and degassing nitrogen gas sealing was performed. And then reacted at 37 ° C. for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0137]
Example 75
5 mg of Met-PTH (1-34) with methionine added to the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4 M urea, and 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM copper sulfate, 4 M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of a solution of 4M acetic acid and 4M sodium acetate containing 80 mM 3,4-diaminobenzoic acid was added, and degassed nitrogen gas sealing was performed. Then, it reacted at 37 degreeC for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0138]
Example 76
5 mg of Met-PTH (1-34) with methionine added to the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 4 M urea, and 4 M sodium acetate, 20 mM acetic acid, 0.4 M glyoxylic acid, 20 mM copper sulfate, 4 M The mixture was mixed with 1 ml of urea solution and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM 2,3-diaminophenol was added, and after deaeration nitrogen gas sealing was performed. , Reacted at 37 ° C. for 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0139]
Example 77
5 mg of Met-PTH (1-34) added with methionine at the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 3 M urea-20 mM Tris / HCl (pH 8.0), and 1 M imidazole, 0.5 M glyoxylic acid , 20 mM copper sulfate and 1 ml of 2.5 M urea solution were mixed and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34). Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM o-phenylenediamine was added, and after degassing nitrogen gas sealing, 37 ° C. For 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0140]
Example 78
5 mg of Met-PTH (1-34) added with methionine at the N-terminus obtained in Reference Example 11 was dissolved in 1 ml of 3 M urea-20 mM Tris / HCl (pH 8.0), and 4 M sodium acetate, 0.8 M acetic acid was dissolved. , 0.4 M glyoxylic acid, 10 mM copper sulfate and 1 ml of 2.5 M urea solution were mixed and reacted at room temperature for 1 hour. After completion of the reaction, centrifugation was performed, and the supernatant was purified by high performance liquid chromatography using an ODP-50 column in the same manner as in Example 66 to obtain a diketone fraction of Met-PTH (1-34).
[0141]
Next, this fraction was freeze-dried, dissolved in 4 ml of distilled water, 4 ml of 4M acetic acid and 4M sodium acetate solution containing 80 mM o-phenylenediamine was added, and after degassing nitrogen gas sealing, 37 ° C. For 17 hours. After completion of the reaction, the product was purified by the same method as in Example 66 to obtain a lyophilized powder of PTH (1-34).
[0142]
【The invention's effect】
According to the present invention, it is possible to selectively and efficiently remove only the methionine residue from a peptide, protein or salt thereof having a methionine residue which may be oxidized at the N-terminus. In addition, according to the method of the present invention, the N-terminal methionine residue can be chemically removed under mild conditions regardless of the type of peptide or protein, and thus the method was produced by genetic engineering techniques. A protein having a natural amino acid sequence can be industrially advantageously produced from a protein to which methionine is added as a raw material.
[0143]
[Sequence Listing]

[0144]

[0145]

[0146]

[Brief description of the drawings]
FIG. 1 shows the result of electrophoresis obtained in Example 2a).
FIG. 2 shows the result of electrophoresis obtained in Example 5a).
FIG. 3 shows the result of electrophoresis obtained in Example 7a).
FIG. 4 shows the result of electrophoresis obtained in Example 9a).
FIG. 5 shows the result of electrophoresis obtained in Example 11a).
FIG. 6 shows the results of electrophoresis obtained in Example 24a).
FIG. 7 shows the results of electrophoresis obtained in Example 38a).
FIG. 8 shows the results of electrophoresis obtained in Example 67a).

Claims (13)

A peptide having a methionine residue which may be oxidized at the N-terminus or a salt thereof and glyoxylic acid or a salt thereof are reacted in the presence of a transition metal ion and a base, and then hydrolyzed. How to remove groups.   The method according to claim 1, wherein the peptide having a methionine residue which may be oxidized at the N-terminus is a peptide produced by genetic engineering.   3. The genetically engineered peptide is a growth hormone, neurotrophin-3, betacellulin, parathyroid hormone or interleukin-2 to which a methionine residue which may be oxidized at the N-terminus is added. the method of.   The process according to claim 1, wherein the base is an alkylamine, an aromatic amine or urea. The method of claim 1 , wherein the transition metal ion is a copper ion. The method of claim 4 , wherein the base is pyridine.   The method according to claim 1, wherein the hydrolysis is carried out using a base. The method according to claim 7 , wherein the base is an amine. The method according to claim 7 , wherein the base is a diamine or thio or selenosemicarbazide. The method according to claim 9, wherein the diamine is o-phenylenediamine.   Peptides selected from human growth hormone, neurotrophin-3, human betacellulin, parathyroid hormone and human interleukin-2, and their salts and glyoxylic acid or its A method for producing the peptide or a salt thereof, which comprises reacting a salt with copper sulfate and pyridine and then reacting with o-phenylenediamine. Formula CH ₃ —S (O) m— (CH ₂ ) ₂ —CO—CO—X [wherein m represents an integer of 0 to 2, and X represents a peptide chain having 2 or more amino acids. Or a salt thereof. A method for producing a peptide or a salt thereof, which comprises hydrolyzing the compound according to claim 12 .

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