JP4512222B2 - Betacellulin variant - Google Patents

Description

【００５４】
【表２】

【００５５】
ｃ）Ｎ末端アミノ酸配列分析
Ｎ末端アミノ酸配列分析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、いずれの改変体もｃＤＮＡ塩基配列から予想されるアミノ酸組成と一致した。いずれの改変体も８０残基型と同様に開始コドンＡＴＧに由来するメチオニンをＮ末端に有していた〔表３，表４〕。
【００５６】
【表３】

【００５７】
【表４】

【００５８】
ｄ）Ｃ末端アミノ酸分析
気相ヒドラジン分解法（１００℃、６時間）により、Ｃ末端アミノ酸をアミノ酸分析計（日立Ｌ−８５００ＡＡｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いて決定した。Ｍｅｔ−７７残基型ベータセルリンではアスパラギン酸が、Ｍｅｔ−７６残基型ベータセルリンではバリンが検出された〔表５，表６〕。
【表５】

【表６】

【００５９】
実施例８
３Ｔ３細胞を用いた増殖促進活性の測定
モレキュラー・セル・バイオロジー、８巻、５８８（１９８８）に記載のごとく、静止状態の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（１９７３））中への³Ｈ−チミジンの取り込みによって増殖促進活性を測定した。
すなわち、５％ ウシ血清を含むダルベッコ改変イーグルＭＥＭ培地を用いて、１０００細胞／ｍＬになるように懸濁した３Ｔ３ Ａ３１−７１４クローン４を９６ウェルプレートに１００μＬづつ播き、炭酸ガスインキュベーター内（５％炭酸ガス、９５％空気）において３７℃で１日培養した。上清７５μＬを抜き取り、血清を含まないダルベッコ改変イーグルＭＥＭ培地を１００μＬ加える事により血清濃度を１％にした。さらに２日間培養した後、種々の濃度の実施例３で調製したＭｅｔ−７７残基型ベータセルリン、実施例６で調製したＭｅｔ−７６残基型ベータセルリン及び特開平１０−１９１９８９号に記載の方法により調製したＭｅｔ−８０残基型ベータセルリンを添加した。添加１６時間後に³Ｈ−チミジン［アマーシャム・ファルマシア・バイオテク］を０．２５μＣｉ／ｗｅｌｌ加え、その４時間後に細胞をＰＢＳで３回洗浄した後、５％ ＳＤＳを１００μＬ加え細胞を溶解した。細胞溶解液をシンチレーションバイアルに移し、シンチレーターＡ（和光純薬社）を１ｍＬ加え、シンチレーションカウンターで³Ｈ−チミジンの細胞への取り込みを測定した〔図４〕。
【００６０】
実施例９
ＡＲ４２Ｊ細胞を用いたβ細胞分化促進活性の測定
化学発癌剤で誘発された膵臓癌由来細胞株ＡＲ４２Ｊ［Ｃｈｉｒｉｓｔｏｐｈｅ；アメリカン・ジャーナル・オブ・フィジオロギー（Ａｍ．Ｊ．Ｐｈｙｓｉｏｌ．），２６６：Ｇ９６３（１９９４）］を、実施例３で調製したＭｅｔ−７７残基型ベータセルリン、実施例６で調製したＭｅｔ−７６残基型ベータセルリンまたは特開平１０−１９１９８９号に記載の方法により調製したＭｅｔ−８０残基型ベータセルリン及び１０％ウシ胎児血清を含むダルベッコ改変イーグルＭＥＭ培地を用いて１０⁵細胞／ｍＬになるように懸濁し、５００μＬをチャンバースライドに播き、炭酸ガスインキュベーター内（５％ 炭酸ガス、９５％空気）において３７℃で５日間培養した。５日後に細胞をＰＢＳで１回洗浄した後に、１０％ホルムアルデヒドで固定し、０．１％ トライトンＸ−１００で５分間処理した後に、ブロックエース（雪印、日本）を添加し室温で４０分間ブロッキングを行った。１０％ ブロックエースで希釈した抗インスリン抗体（アドバンスド・イムノ・ケミカル社）を添加し室温、４０分間反応した。０．１％ トライトンＸ−１００を添加し室温５分間放置した後、ＰＢＳで３回洗浄した。１０％ブロックエースで希釈したＦＩＴＣ（Ｆｌｕｏｒｅｓｃｅｉｎ Ｉｓｏｔｈｉｏｃｙａｎａｔｅ）標識抗マウスＩｇＧ抗体（カッペル社）を添加し、室温で４０分間反応した。０．１％トライトンＸ−１００を添加し室温５分間放置した後、ＰＢＳで３回洗浄し、蛍光顕微鏡で観察した。いずれのベータセルリンを添加した場合においても、蛍光染色された細胞、すなわちインスリンを産生するβ細胞に分化した細胞が観察された〔図５〕。
【００６１】
実施例１０
ヒト胎盤アルカリフォスファターゼ遺伝子発現ベクターの構築
β細胞への分化促進活性はインスリンプロモーターの下流にレポーターとしてアルカリフォスファターゼを連結したものにより形質転換されたＡＲ４２Ｊ細胞を用いても実施した。すなわちベーターセルリンによりβ細胞へと分化した細胞はアルカリフォスファターゼを産生し、このアルカリフォスファターゼの活性を測定することによりβ細胞への分化促進活性を定量的に測定することが可能である。
ラット尾から常法に従ってゲノムＤＮＡを調製した。このＤＮＡをテンプレートとして、既報のラットインスリンＩＩ遺伝子プロモーターの塩基配列（ＧｅｎＢａｎｋ：Ａｃｃｅｓｓｉｏｎ Ｎｏ．Ｊ００７４８）をもとに合成したプライマーＲＩ−１［５’−ＡＧＡＧＴＣＡＡＧＧＡＴＣＣＣＣＣＡＡＣＣＡＣＴ−３’］およびＲＩ−３［５’−ＡＧＣＴＧＧＴＣＡＣＴＴＡＧＧＧＣＴＧＧＧＧ−３’］を用いてＰＣＲ法によりインスリンプロモーター領域０．７５Ｋｂを増幅した。さらに、このＰＣＲ産物をテンプレートとしてプライマーＲＩ−１Ｃｌａ［５’−ＧＡＡＴＣＧＡＴＡＧＡＧＴＣＡＡＧＧＡＴＣＣＣＣＣＡ−３’］およびＲＩ−３Ｘｈｏ［５’−ＧＡＣＴＣＧＡＧＣＴＧＧＴＣＡＣＴＴＡＧＧＧ−３’］を用いてＰＣＲを行った。増幅された０．７５Ｋｂ ＤＮＡ断片を単離し、ｐＴ７Ｂｌｕｅベクター（Ｎｏｖａｇｅｎ ６９８２０−１）に組み込んで得られたプラスミドｐＴＢ１８８１を用いてクローニングされた断片の塩基配列を決定し、ラットインスリンプロモーターであることを確認した。プラスミドｐＴＢ１８８１をＸｈｏ Ｉ−Ｃｌａ Ｉで切断してラットインスリンプロモーターである０．７３ｋｂ ＤＮＡ断片を得た。次にヒト胎盤アルカリフォスファターゼ（ＰＬＡＰ）をコードする２．０ ｋｂ ｃＤＮＡ（Ｊ．Ｂｅｒｇｅｒら、Ｇｅｎｅ ６６，１（１９８８））の発現プラスミドｐＴＢ１３３０をＸｈｏ Ｉ−Ｈｉｎｄ ＩＩＩで切断して得られた２．７ｋｂ ＤＮＡ断片（ＰＬＡＰｃＤＮＡ、ＳＶ４０由来スプライシング部位およびポリＡ付加部位、ｐＢＲ３２２由来ｏｒｉおよびアンピシリン耐性遺伝子を含む）を単離し、前述のラットインシュリンプロモーター領域０．７３ｋｂ Ｘｈｏ Ｉ−Ｃｌａ Ｉ断片をＴ４ ＤＮＡリガーゼ反応によって連結してプラスミドｐＴＢ１８９８を得た。
【００６２】
実施例１１
ＰＬＡＰ発現ＡＲ４２Ｊ細胞の構築
ＡＲ４２Ｊ細胞にＰＬＡＰ発現プラスミドｐＴＢ１８９８とＴｎ５のｎｅｏ^r遺伝子を含むプラスミドｐＭＣｎｅｏｐｏｌｙ Ａ（ＳＴＲＡＴＡＧＥＮＥ）をトランスフェクション試薬ＴｒａｎｓＩＴ^TM−ＬＴ１（Ｍｉｒｕｓ、ＰｅｎＶｅｒａ Ｃｏｒｐｏｒａｔｉｏｎ）を用いて同時に導入した。導入後の細胞は１０％牛胎児血清を加えたＤＭＥＭで２日間培養した後、Ｇ４１８（ゲネテシン、ＧＩＢＣＯ ＢＲＬ）８００ μｇ／ｍＬを添加した選択培地に加えて培養を続けた。Ｇ４１８耐性となって生育した細胞を限界希釈法によりクローンを単離した。
各クローンの細胞を２４穴プレートに播き、Ｍｅｔ−８０残基型ベータセルリンを２０ ｎｇ／ｍＬ添加および無添加で４日間培養し、培養上清を集めて６５℃３０分間熱処理を行った後、培地中のアルカリフォスファターゼ活性を測定した。８０残基型ベータセルリン添加時にアルカリフォスファターゼ活性の上昇が認められるクローンを選択した。いくつかのクローンの結果を以下の〔表７〕に示す。
【表７】

【００６３】
実施例１２
ＰＬＡＰ発現ＡＲ４２Ｊ細胞を用いたβ細胞分化促進活性の測定
実施例１１で構築したＰＬＡＰ発現ＡＲ４２Ｊ細胞を、各種濃度の実施例３で調製したＭｅｔ−７７残基型ベータセルリン、実施例６で調製したＭｅｔ−７６残基型ベータセルリンまたは特開平１０−１９１９８９号に記載の方法により調製したＭｅｔ−８０残基型ベータセルリン及び１０％ウシ胎児血清を含むダルベッコ改変イーグルＭＥＭ培地を用いて１０⁵細胞／ｍＬになるように懸濁し、１００μＬを９６ウェルプレートに播き、炭酸ガスインキュベーター内（５％ 炭酸ガス、９５％ 空気）において３７℃で５日間培養した。５日後に培養上清をサンプリングし、６５℃で３０分間処理した後、５０μＬをあらかじめ５０μＬの２ＸＳＥＡＰ（２Ｍ ジエタノールアミン、１ｍＭ ＭｇＣｌ₂、２０ｍＭ ホモアルギニン）を添加した９６ウェルマイクロプレートに加えた。３７℃で１０分間保った後、２０ｍｇ／ｍＬのｐ−ニトロフェニルリン酸（シグマ社）を１０ μＬ添加し３７℃で１６時間反応を行った〔図６〕。Ｍｅｔ−７７残基型ベータセルリンおよびＭｅｔ−７６残基型ベータセルリンはＭｅｔ−８０残基型ベータセルリンとほぼ同じ分化誘導促進活性を示した。
【００６４】
実施例１３
２−７６残基型（Ｎ末端１残基、Ｃ末端３残基欠失型）ベータセルリン発現株の構築
２−７６残基型（Ｎ末端１残基、Ｃ末端３残基欠失型）ベータセルリンの発現プラスミドは以下のように構築した〔図７〕。
２−７６残基型（Ｎ末端１残基、Ｃ末端３残基欠失型）ベータセルリンの構造遺伝子を、７６残基型（Ｃ末端３残基欠失型）ベータセルリン発現プラスミドｐＴＣＩＩＢＴＣ７６［実施例４］より、２番目のグリシンの上流に隣接してＮｄｅ Ｉ切断部位及び開始コドンを持つプライマー３（５'−ＣＡＧＣＡＴＡＴＧＧＧＧＡＡＴＴＣＣＡＣＣＡＧＡＡＧＴＣＣＴ）、およびＣ末端のバリンの後に終止コドン及びＢａｍ ＨＩ切断部位を持つプライマー４（５' −ＧＧＡＴＣＣＣＴＡＡＡＣＴＣＴＣＴＣＡＣＡＣＣＴＴＧＣＴＣＣＡＡＴＧ）を用いて、ＰＣＲで増幅した。ＰＣＲにより増幅した遺伝子を、ＴＡ ｏｒｉｇｉｎａｌ ｃｌｏｎｉｎｇ ｋｉｔ（インヴィトロジェン社製）を用いてｐＣＲ２．１ベクターに連結し、ｐＣＲ２．１ＢＴＣ２−７６を作製した。これを大腸菌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。ｐＣＲ２．１ＢＴＣ２−７６を有する形質転換体を培養し、ＱＩＡｐｒｅｐ８ Ｍｉｎｉｐｒｅｐ ｋｉｔ（キアゲン社製）を用いてｐＣＲ２．１ＢＴＣ２−７６を調製した。
ｐＣＲ２．１ＢＴＣ２−７６をＮｄｅ Ｉ及びＢａｍ ＨＩで切断してアガロース電気泳動を行い、約２３０ ｂｐの２−７６残基型ベータセルリン構造遺伝子をＱＩＡＧＥＮ ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（キアゲン社製）を用いて回収した。発現ベクターｐＴＣＩＩ［実施例１］をＮｄｅ Ｉ及びＢａｍ ＨＩで切断してアガロース電気泳動を行い、同様に約４．６ｋｂｐのバンドを回収した。２−７６残基型ベータセルリン構造遺伝子を発現ベクターｐＴＣＩＩのＮｄｅ Ｉ−Ｂａｍ ＨＩ断片と連結した後、大腸菌ＪＭ１０９に導入してテトラサイクリン耐性で形質転換株を選択し、その株より再度プラスミドを回収して、発現プラスミドｐＴＣＩＩＢＴＣ２−７６とした。
このｐＴＣＩＩＢＴＣ２−７６を大腸菌ＭＭ２９４（ＤＥ３）に導入して、テトラサイクリン耐性で形質転換株を選択し、２−７６残基型ベータセルリン発現株ＭＭ２９４（ＤＥ３）／ｐＴＣＩＩＢＴＣ２−７６を取得した。
【００６５】
実施例１４
２−７６残基型ベータセルリン発現株の培養
２−７６残基型ベータセルリン発現株ＭＭ２９４（ＤＥ３）／ｐＴＣＩＩＢＴＣ２−７６を１０ｍｇ／Ｌのテトラサイクリンを含むＬＢ培地（１％ ペプトン、０．５％ 酵母エキス、０．５％ 塩化ナトリウム）３０ｍＬで３０℃、１６時間培養した。得られた培養液を主発酵用培地（１．６８％ リン酸一水素ナトリウム、０．３％ リン酸二水素ナトリウム、０．１％ 塩化アンモニウム、０．０５％ 塩化ナトリウム、０．０１２％ 硫酸マグネシウム、０．０００７％ 塩酸チアミン、１．５％ ブドウ糖、１．５％ ハイケース）２００ｍＬを入れた１Ｌ容マイヤー６本に１０ｍｌずつ移植して、３７℃、撹拌回転数２００ｒｐｍで撹拌培養を開始した。培養液の濁度が約１６０クレット単位になった時点で、５．９５ ｍｇ／Ｌ分のイソプロピル−β−Ｄーチオガラクトピラノシド（ＩＰＴＧ）を添加した。ＩＰＴＧ添加後４時間後まで培養を行った。培養液を９０００ｒｐｍで３０分間遠心分離を行い、菌体５．１ｇを集めた。
【００６６】
実施例１５
２−７６残基型ベータセルリンの精製
実施例１４で得られた菌体５ｇに１ｍＭ ＥＤＴＡ、１ｍＭ ＡＰＭＳＦ及び７Ｍ グアニジン塩酸塩を含む０．１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）１５ｍＬを加え、４℃で一晩撹拌し抽出を行った後、遠心分離（９０００ｒｐｍ、１０分間）を行った。得られた上清液１５ｍＬに０．５ｍＭ 酸化型グルタチオン、１ｍＭ 還元型グルタチオン、１ｍＭ ＥＤＴＡ、０．１Ｍ アルギニン塩酸塩及び２Ｍ 尿素を含む５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）２５０ｍＬを加え、４℃で一晩リフォールディングを行った後、遠心分離（１００００ｒｐｍ、１０分間）を行い、遠心上清液２６５ｍＬを得た。この遠心上清液に２Ｍ 尿素を１Ｌ加えた後、酢酸でｐＨ５．０に調整し、４℃で一晩静置した。遠心分離（１００００ｒｐｍ、１０分間）を行い、得られた遠心上清液を５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．０）で平衡化したＰＯＲＯＳ ５０ＨＳカラム（２．２ｃｍ×１２ｃｍ、日本パーセプティブ社）に毎分３０ｍＬの流速で吸着させ、平衡化に用いた緩衝液でよく洗浄した後、０．３Ｍから１．３Ｍの塩化ナトリウム直線濃度勾配により溶出を行った。２−７６残基型ベータセルリンを含む画分を集め、５０ ｍＭ酢酸ナトリウム緩衝液（ｐＨ４．５）で３倍に希釈した後、この緩衝液で平衡化したＴＳＫｇｅｌ ＣＭ−５ＰＷカラム（０．７５ｃｍ×７．５ｃｍ、東ソー社）に添加した。２−７６残基型ベータセルリンが吸着したＴＳＫｇｅｌ ＣＭ−５ＰＷカラムを、０．３Ｍから０．５Ｍの塩化ナトリウム直線濃度勾配により溶出を行った。２−７６残基型ベータセルリンを含む画分を集め、０．１％ トリフルオロ酢酸で平衡化したＴＳＫｇｅｌ ＯＤＳ−１２０Ｔカラム（２．１５ｃｍ×３０ｃｍ、東ソー社）に吸着させ、２０％から４４％のアセトニトリル直線濃度勾配により２−７６残基型ベータセルリンを溶出した。溶出液を凍結乾燥した後、蒸留水５ｍＬで溶解し、酢酸型にしたＡＧ１−Ｘ８カラム（１．０ｃｍ×１０ｃｍ、日本バイオラッド社）を通過させた後、再度凍結乾燥を行い２−７６残基型ベータセルリン０．７ｍｇを得た。
【００６７】
実施例１６
２４−７６残基型（Ｎ末端２３残基、Ｃ末端３残基欠失型）ベータセルリン発現株の構築
２４−７６残基型（Ｎ末端２３残基、Ｃ末端３残基欠失型）ベータセルリンの発現プラスミドは以下のように構築した〔図８〕。
２４−７６残基型（Ｎ末端２３残基、Ｃ末端３残基欠失型）ベータセルリンの構造遺伝子を、実施例４で調製した７６残基型（Ｃ末端３残基欠失型）ベータセルリン発現プラスミドｐＴＣＩＩＢＴＣ７６より、２４番目のアラニンの上流に隣接してＮｄｅ Ｉ切断部位及び開始コドンを持つプライマー５（５’−ＣＡＧＣＡＴＡＴＧＧＣＴＡＣＣＡＣＣＡＣＡＣＡＡＴＣＡＡＡＧ）、およびＣ末端のバリンの後に終止コドン及びＢａｍ ＨＩ切断部位を持つプライマー４（５’−ＧＧＡＴＣＣＣＴＡＡＡＣＴＣＴＣＴＣＡＣＡＣＣＴＴＧＣＴＣＣＡＡＴＧ）を用いて、ＰＣＲで増幅した。ＰＣＲにより増幅した遺伝子を、ＴＡ ｏｒｉｇｉｎａｌ ｃｌｏｎｉｎｇ ｋｉｔ（インヴィトロジェン社製）を用いてｐＣＲ２．１ベクターに連結し、ｐＣＲ２．１ＢＴＣ２４−７６を作製した。これを大腸菌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。ｐＣＲ２．１ＢＴＣ２４−７６を有する形質転換体を培養し、ＱＩＡｐｒｅｐ８ Ｍｉｎｉｐｒｅｐ ｋｉｔ（キアゲン社製）を用いてｐＣＲ２．１ＢＴＣ２４−７６を調製した。
ｐＣＲ２．１ＢＴＣ２４−７６をＮｄｅ Ｉ及びＢａｍ ＨＩで切断してアガロース電気泳動を行い、約１６０ｂｐの２４−７６残基型ベータセルリン構造遺伝子をＱＩＡＧＥＮ ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（キアゲン社製）を用いて回収した。実施例１で調製した発現ベクターｐＴＣＩＩをＮｄｅ Ｉ及びＢａｍ ＨＩで切断してアガロース電気泳動を行い、同様に約４．６ｋｂｐのバンドを回収した。２４−７６残基型ベータセルリン構造遺伝子を発現ベクターｐＴＣＩＩのＮｄｅ Ｉ−Ｂａｍ ＨＩ断片と連結した後、大腸菌ＪＭ１０９に導入してテトラサイクリン耐性で形質転換株を選択し、その株より再度プラスミドを回収して、発現プラスミドｐＴＣＩＩＢＴＣ２４−７６とした。
このｐＴＣＩＩＢＴＣ２４−７６を大腸菌ＭＭ２９４（ＤＥ３）に導入して、テトラサイクリン耐性で形質転換株を選択し、２４−７６残基型ベータセルリン発現株ＭＭ２９４（ＤＥ３）ｐＴＣＩＩＢＴＣ２４−７６を取得した。
【００６８】
実施例１７
２４−７６残基型ベータセルリン発現株の培養
２４−７６残基型ベータセルリン発現株ＭＭ２９４（ＤＥ３）／ｐＴＣＩＩＢＴＣ２４−７６を１０ｍｇ／Ｌのテトラサイクリンを含むＬＢ培地（１％ ペプトン、０．５％ 酵母エキス、０．５％ 塩化ナトリウム）３０ｍＬで３０℃、１６時間培養した。得られた培養液を主発酵用培地（１．６８％ リン酸一水素ナトリウム、０．３％ リン酸二水素ナトリウム、０．１％ 塩化アンモニウム、０．０５％ 塩化ナトリウム、０．０１２％ 硫酸マグネシウム、０．０００７％ 塩酸チアミン、１．５％ ブドウ糖、１．５％ ハイケース）１５０ｍＬを入れた１Ｌ容マイヤー５本に移植して、３７℃、撹拌回転数２００ｒｐｍで撹拌培養を開始した。培養液の濁度が約１６０クレット単位になった時点で、５．９５ｍｇ／Ｌ分のイソプロピル−β−Ｄ−チオガラクトピラノシド（ＩＰＴＧ）を添加した。ＩＰＴＧ添加後４時間後まで培養を行った。培養液を１００００ｒｐｍで３０分間遠心分離を行い、菌体３．９ｇを集めた。
【００６９】
実施例１８
２４−７６残基型ベータセルリンの精製
実施例５で得られた菌体３．９ｇに１ｍＭ ＥＤＴＡ、１ｍＭ ＡＰＭＳＦ及び７Ｍ グアニジン塩酸塩を含む０．１Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）１２ｍＬを加え、４℃で一晩撹拌し抽出を行った後、遠心分離（９０００ｒｐｍ、１０分間）を行った。得られた上清液１２ｍＬに０．５ｍＭ 酸化型グルタチオン、１ｍＭ 還元型グルタチオン、１ｍＭ ＥＤＴＡ、０．１Ｍ アルギニン塩酸塩及び２Ｍ 尿素を含む５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）２００ｍＬを加え、４℃で一晩リフォールディングを行った後、遠心分離（１００００ｒｐｍ、１０分間）を行い、遠心上清液２１２ｍＬを得た。この遠心上清液に２Ｍ尿素を０．８Ｌ加えた後、酢酸でｐＨ５．０に調整し、４℃で一晩静置した。遠心分離（１００００ｒｐｍ、１０分間）を行い、得られた遠心上清液を５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．０）で平衡化したＰＯＲＯＳ ５０ＨＳカラム（２．２ｃｍ×１２ｃｍ、日本パーセプティブ社）に毎分３０ｍＬの流速で吸着させ、平衡化に用いた緩衝液でよく洗浄した後、０．３Ｍから１．３Ｍの塩化ナトリウム直線濃度勾配により溶出を行った。２４−７６残基型ベータセルリンを含む画分を集め、５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ４．５）で３倍に希釈した後、この緩衝液で平衡化したＴＳＫｇｅｌ ＣＭ−５ＰＷカラム（０．７５ｃｍ×７．５ｃｍ、東ソー社）に添加した。２４−７６残基型ベータセルリンが吸着したＴＳＫｇｅｌ ＣＭ−５ＰＷカラムを、０．３Ｍから０．５Ｍの塩化ナトリウム直線濃度勾配により溶出を行った。２４−７６残基型ベータセルリンを含む画分を集め、０．１％ トリフルオロ酢酸で平衡化したＴＳＫｇｅｌ ＯＤＳ−１２０Ｔカラム（２．１５ｃｍ×３０ｃｍ、東ソー社）に吸着させ、２０％から４４％のアセトニトリル直線濃度勾配により２４−７６残基型ベータセルリンを溶出した。溶出液を凍結乾燥した後、蒸留水５ｍＬで溶解し、酢酸型にしたＡＧ１−Ｘ８カラム（１．０ｃｍ×１０ｃｍ、日本バイオラッド社）を通過させた後、再度凍結乾燥を行い２４−７６残基型ベータセルリン０．４ｍｇを得た。
【００７０】
実施例１９
ベータセルリン改変体の特徴の決定
実施例３で得られた２−７６残基型ベータセルリン及び実施例６で得られた２４−７６残基型ベータセルリンの特徴を以下のように決定した。
ａ）ＳＤＳ−ポリアクリルアミド電気泳動を用いた分析
ベータセルリン改変体及び実施例６で調製した７６残基型ベータセルリンをサンプルバッファー（１２５ｍＭ Ｔｒｉｓ−ＨＣｌ、１％ ドデシル硫酸ナトリウム、１５％ グリセロール、５％ ２−メルカプトエタノール、０．００５％ ブロムフェノールブルー）で懸濁し、マルチゲル１５／２５（第一化学薬品）で電気泳動を行った。泳動後のゲルをラピッド ＣＢＢ ＫＡＮＴＯ（関東化学社）で染色を行ったところ、いずれもほぼ単一バンドであった〔図９〕。
ｂ）アミノ酸組成分析
ベータセルリン改変体を４％チオグリコール酸を含む６ Ｎ塩酸で１１０℃、２４及び４８時間気相加水分解を行い、アミノ酸分析計（日立Ｌ−８５００Ａ Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いてアミノ酸組成を決定した。その結果、いずれの改変体もｃＤＮＡ塩基配列から予想されるアミノ酸組成と一致した〔表８，表９〕。
【００７１】
【表８】

【００７２】
【表９】

【００７３】
ｃ）Ｎ末端アミノ酸配列分析
Ｎ末端アミノ酸配列分析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、いずれの改変体もｃＤＮＡ塩基配列から予想されるアミノ酸配列と一致した〔表１０，表１１〕。
【００７４】
【表１０】

【００７５】
【表１１】

【００７６】
ｄ）Ｃ末端アミノ酸分析
気相ヒドラジン分解法（１００℃、６時間）により、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。いずれの改変体もバリンが検出された〔表１２，表１３〕。
【表１２】

【表１３】

【００７７】
実施例２０
１）Ａ４３１細胞を用いたＥＧＦバインディングアッセイ
ＥＧＦレセプター高発現株であるヒト扁平上皮癌細胞Ａ４３１細胞を用いた¹²⁵Ｉ−ＥＧＦに対する結合阻害活性によって細胞増殖促進活性を測定した。
すなわち、１０％ ウシ胎児血清を含むダルベッコ改変イーグルＭＥＭ（ＤＭＥＭ）培地を用いて、１０⁵細胞／ｍＬになるように懸濁したＡ４３１細胞を９６ウェルプレートに１００μＬずつ播き、炭酸ガスインキュベーター内（５％炭酸ガス、９５％空気）において３７℃で２日間培養した。２日後に２０ｍＭ ＨＥＰＥＳおよび０．１％ ウシ血清アルブミンを含む、ＤＭＥＭとＦ−１２を等量混合した結合培地２００μＬ／ウェルで３回洗浄した後、各種濃度の標識されていないＥＧＦ、実施例１５で調製した２−７６残基型ベータセルリン、実施例１８で調製した２４−７６残基型ベータセルリン、実施例６で調製したＭｅｔ−７６残基型ベータセルリン、または特開平１０−１９１９８９号に記載の用法により調製したＭｅｔ−８０残基型ベータセルリン及び０．２５ｎＭ ¹²⁵Ｉ−ＥＧＦ（アマシャム・ファルマシア・バイオテク）を含む結合培地１００μＬを添加した。４℃、９０分静置した後に結合培地２００μＬで３回洗浄し、１％ ＳＤＳを含む０．１Ｎ 水酸化ナトリウム水溶液２００μＬで細胞を溶解した。細胞溶解液をシンチレーションバイアルに移し、シンチレーターＡ（和光純薬社）を１ｍＬ加え、シンチレーションカウンターで¹²⁵Ｉ−ＥＧＦに対する結合阻害活性を測定した。
２）ヒト胎盤アルカリフォスファターゼ発現ＡＲ４２Ｊ細胞を用いたβ細胞分化促進活性の測定
ＰＬＡＰ発現ＡＲ４２Ｊ細胞を、各種濃度の実施例１５で調製した２−７６残基型ベータセルリン、実施例１８で調製した２４−７６残基型ベータセルリン、実施例６で調製したＭｅｔ−７６残基型ベータセルリン、または特開平１０−１９１９８９号に記載の用法により調製したＭｅｔ−８０残基型ベータセルリン及び１０％ ウシ胎児血清を含むダルベッコ改変イーグルＭＥＭ培地を用いて１０⁵細胞／ｍＬになるように懸濁し、１００μＬを９６ウェルプレートに播き、炭酸ガスインキュベーター内（５％炭酸ガス、９５％ 空気）において３７℃で５日間培養した。５日後に培養上清をサンプリングし、６５℃で３０分間処理した後、５０μＬをあらかじめ５０μＬの２ｘＳＥＡＰ（２Ｍ ジエタノールアミン、１ｍＭ ＭｇＣｌ₂、２０ｍＭ ホモアルギニン）を添加した９６ウェルマイクロプレートに加えた。２０ｍｇ／ｍＬのｐ−ニトロフェニルリン酸（シグマ社）を１０μＬ添加し３７℃で１６時間反応を行った。いずれの改変体もＥＧＦ活性対ＢＴＣ活性の比がＭｅｔ−８０残基型ベータセルリンの５％ほどに低下し、Ｍｅｔ−７６残基型ベータセルリンと同程度でありＮ末欠失による影響は認められなかった。
３)上記１）および２）の結果を〔表１４〕にまとめ示す。
【表１４】

【００７８】
実施例２１
Ｃｙｓ３−Ｃｙｓ４間にヘレギュリン（Ｈｅｒ）に由来する３残基が挿入されたｈＢＴＣ５０改変分子Ａ発現プラスミドｐＴＢ１９８５の構築
ｐＴＢ１９７６をテンプレートとして、（１）５'側プライマーＰＥＴ−１：５'−ＧＡＡＡＴＡＡＴＴＴＴＧＴＴＴＡＡＣＴＴＴＡＡＧＡＡＧＧＡＧ−３'および３'側プライマーＢＴＣ−１：５'−ＡＧＧＡＧＧＧＣＧＴＣＧＡＧＧＧＧＴＴＣＴＧＣＴＣＧＧＣＣＡ−３'、（２）５'側プライマーＢＴＣ−２：５'−ＴＧＧＣＣＧＡＧＣＡＧＡＡＣＣＣＣＴＣＧＡＣＧＣＣＣＴＣＣＴ−３'および３'側プライマーＢＴＣ−３：５'−ＴＣＴＡＴＧＣＧＣＡＣＣＣＧＴＴＣＴＣＧＧＡＧＣＡＣＴＧＴＣ−３'を用いて、それぞれＰＣＲを行った。
次に（１）と（２）のＰＣＲ産物の混合物をテンプレートとして、５'側プライマーＢＴ−９５ｈ、３’側プライマーＢＴ−９４ｈを用いてＰＣＲを行い、ｈＢＴＣ５０のＣｙｓ３−Ｃｙｓ４間にＨｅｒの配列中の３アミノ酸（Ａｓｎ，Ｐｒｏ，Ｓｅｒ）を挿入した改変分子ＡをコードするＤＮＡ断片を得た。このＤＮＡ断片をＮｄｅＩおよびＢａｍＨＩで消化後、ｐＥＴ−３ｃのＮｄｅＩ−ＢａｍＨＩ部位に組み込み、ｐＴＢ１９８５を作製した。
【００７９】
実施例２２
Ｎ末７残基をその部位に相当する上皮成長因子（ＥＧＦ）の５残基で置換されたｈＢＴＣ５０改変分子Ｂ発現プラスミドｐＴＢ１９８７の構築
ｐＴＢ１９７６をテンプレートとして、５'側プライマーＢＴＣ−７：５'−ＴＡＴＡＣＡＴＡＴＧＡＡＣＡＧＣＧＡＣＴＣＴＧＡＧＴＧＣＣＣＣＡＡＧＣ−３'および３'側プライマーＢＴ−９４ｈを用いてＰＣＲを行い、ｈＢＴＣ５０のＮ末７残基を相当するＥＧＦの５残基に置換した改変分子ＢをコードするＤＮＡ断片を得た。このＤＮＡ断片をＮｄｅＩおよびＢａｍＨＩで消化後、ｐＥＴ３ｃのＮｄｅＩ−ＢａｍＨＩ部位に組み込み、ｐＴＢ１９８７を作製した。
【００８０】
実施例２３
大腸菌での発現
Ｔ７ファージのΦ１０プロモーターの支配下に、後述の参考例および上記の実施例で得られたプラスミドｐＴＢ１９７６、プラスミドｐＴＢ１９８５およびプラスミドｐＴＢ１９８７を、発現用大腸菌ＢＬ２１（ＤＥ３）／ｐＬｙｓＳ（Ｎｏｖａｇｅｎ）に導入した。それぞれの大腸菌組換え体を１００μｇ／ｍｌアンピシリンおよび１０μｇ／ｍｌクロラムフェニコールを含むＬＢ培地を用いて３７℃で培養した。クレットユニット１６０の時点でイソプロピル β−Ｄ−チオガラクトピラノシドを最終濃度０．４ｍＭになるように添加し、さらに３７℃で４時間培養を継続した。培養後遠心により集菌し、抽出操作まで−８０℃で保存した。
【００８１】
実施例２４
ｈＢＴＣ５０、改変分子Ａおよび改変分子Ｂの精製
４−１）大腸菌封入体の調製
４００ｍｌの培養液から集めた菌体を２０ｍｌの１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、１０％ ｓｕｃｒｏｓｅ、１０ｍＭ ＥＤＴＡに懸濁して、凍結・融解した後、氷中に３０分間静置して完全に細胞を溶かした。氷冷下で超音波破砕（１０ｓｅｃ，３回）後、遠心（９０００ｒｐｍ，１５ｍｉｎ，４℃：Ｂｅｃｋｍａｎ）して再度沈殿を集めた。この洗浄操作を３回繰り返して封入体を調製した。
４−２）タンパクの抽出およびリフォールデイング
４−１）で得られた封入体を８ｍｌの７Ｍ ｇｕａｎｉｄｉｎｅ−ＨＣｌ、０．１Ｍ Ｔｒｉｓ−ＣＨ₃ＣＯＯＨ（ｐＨ８．０）、１ｍＭ ＥＤＴＡに懸濁して４℃、１時間スターラーで穏やかに撹拌し、組換えタンパクを抽出した。次に還元型グルタチオンを０．１Ｍになるように添加して、ＮａＯＨ溶液でｐＨを８．４に合わせた後、抽出液中に窒素ガスを吹き込み空気と置換した。室温で２時間静置した後、２０倍容量の１０ｍＭ Ｔｒｉｓ−ＣＨ₃ＣＯＯＨ（ｐＨ８．０）、０．５ｍＭ フェニルメチルスルホニルフルオリド、１ｍＭ ベンズアミジンで稀釈した。遠心（９０００ｒｐｍ，１５ｍｉｎ，２５℃）して不溶物を除いた上清に、酸化型グルタチオンを０．５ｍＭになるように添加し、室温で１６時間静置した。次いで、遠心（９０００ｒｐｍ，１５ｍｉｎ，２５℃）上清を凍結乾燥して濃縮後、４℃で５０ｍＭ Ｔｒｉｓ−ＣＨ₃ＣＯＯＨ（ｐＨ５．５）、１ｍＭＥＤＴＡに透析（Ｓｐｅｃｔｒａ ｐｏｒ：ＭＷＣＯ １，０００）した。透析後、遠心（９０００ｒｐｍ，１５ｍｉｎ，４℃）上清を酢酸セルロース膜（Ｍｉｌｌｅｘ ＧＶ，０．２２ ｕｍ：Ｍｉｌｌｉｐｏｒｅ）で濾過して、不溶物を除いた。
４−３）タンパクの精製
リフォールデイングしたタンパクを含む溶液を５０ｍＭ 酢酸ナトリウム（ｐＨ５．５）で平衡化した陽イオン交換ＨＰＬＣカラム（２５０×４．１ｍｍ Ｉ．ｄ．，Ｓｙｎｃｈｒｏｍ ＣＭ３００，Ｓｙｎｃｈｒｏｐａｋ）にかけ、０−１ＭのＮａＣｌ濃度勾配でタンパクを溶出、分画した。ＯＤ₂₈₀でモニターした溶出画分を、０．１％ ＨＣｌ、１％ ＣＨ₃ＣＮで平衡化したＣ₄逆相ＨＰＬＣカラム（１５０×４．６ｍｍ Ｉ．ｄ．，Ｕｌｔｒｏｎ ３００ Ｃ₄：Ｃｈｒｏｍａｔｏｐａｃｋｉｎｇ Ｃｅｎｔｅｒ）にかけて、１−４０％のＣＨ₃ＣＮ濃度勾配で溶出した。次にカラムクロマトグラフィーで得たピーク画分を凍結乾燥した。改変分子ＡおよびＢの収量は、各々７８．７μｇおよび１０３．１μｇであった。
４−４）タンパクの純度検定
精製標品の純度検定の目的で、Ｃ₁₈逆相ＨＰＬＣカラム（１５０×４．６ｍｍｉ．ｄ．，ＯＤＳ１２０Ｔ，Ｔｏｓｏｈ）にかけて、１５〜３０％のＣＨ₃ＣＮ濃度勾配で溶出し、改変分子ＡおよびＢのピークパターンの単一性を確認した。両タンパクの純度は９４％以上であることが分かった。
【００８２】
実施例２５
３Ｔ３細胞を用いた細胞増殖促進活性の測定
モレキュラー・アンド・セルラー・バイオロジー、8巻、588頁、1988年に記載の方法に準じ、静止状態の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）中への³Ｈ−チミジンの取り込みによって細胞増殖促進活性を測定した。
５％ ウシ血清を含むダルベッコ改変イーグルＭＥＭ培地を用いて、１０００細胞／ｍＬになるように懸濁した３Ｔ３ Ａ３１−７１４クローン４を９６ウェルプレートに１００μＬづつ播き、炭酸ガスインキュベーター内（５％炭酸ガス、９５％空気）において３７℃で１日培養した。上清７５μＬを抜き取り、血清を含まないダルベッコ改変イーグルＭＥＭ培地を１００μＬ加えることにより血清濃度を１％にした。さらに２日間培養した後、種々の濃度の上記の実施例で調製したｈＢＴＣ５０、改変分子Ａまたは改変分子Ｂを添加した。添加１６時間後に³Ｈ−チミジン［アマーシャム・ファルマシア・バイオテク］を０．２５μＣｉ／ｗｅｌｌ加え、その４時間後に細胞をＰＢＳで３回洗浄した後、５％ ＳＤＳを１００μＬ加え細胞を溶解した。細胞溶解液をシンチレーションバイアルに移し、シンチレーターＡ（和光純薬社）を１ｍＬ加え、シンチレーションカウンターで³Ｈ−チミジンの細胞への取り込みを測定した。
この測定結果より、８０残基型ベータセルリン（標準品）の最大取り込み値を１００％として、５０％の活性を示した時のｈＢＴＣ５０および改変分子の濃度（ＥＤ₅₀）を求めた結果を〔表１５〕に示す。
【表１５】

【００８３】
実施例２６
ＡＲ４２Ｊ細胞を用いたβ細胞分化誘導活性の測定
上記で調製したｈＢＴＣ５０、改変分子Ａ、改変分子Ｂおよび特開平１０−１９１９８９号に記載の方法により調製した８０残基型ベータセルリン（ｈＢＴＣ８０）から選ばれる１種と、１０％ ウシ胎児血清とを含むダルベッコ改変イーグルＭＥＭ培地に、化学発癌剤で誘発された膵臓癌由来細胞株ＡＲ４２Ｊ［Chiristophe; アメリカン・ジャーナル・オブ・フィジオロギー(Am. J. Physiol.）,２６６巻、Ｇ９６３頁、１９９４年］を１０⁵細胞／ｍＬになるように懸濁し、５００ μＬをチャンバースライドに播き、炭酸ガスインキュベーター内（５％ 炭酸ガス、９５％ 空気）において３７℃で５日間培養した。５日後に細胞をＰＢＳで１回洗浄した後に、１０％ ホルムアルデヒドで固定し、０．１％ トリトンＸ−１００で５分間処理した後に、ブロックエース（雪印、日本）を添加し、室温で４０分間ブロッキングを行った。１０％ ブロックエースで希釈した抗インスリン抗体（アドバンスド・イムノ・ケミカル社）を添加し室温、４０分間反応した。０．１％ トリトンＸ−１００を添加し室温５分間放置した後、ＰＢＳで３回洗浄した。１０％ ブロックエースで希釈したＦＩＴＣ（Fluorescein Isothiocyanate）標識抗マウスＩｇＧ抗体（カッペル社）を添加し、室温で４０分間反応した。０．１％トリトンＸ−１００を添加し室温５分間放置した後、ＰＢＳで３回洗浄し、蛍光顕微鏡で観察した。
その結果、ｈＢＴＣ８０を添加した場合と同様に、ｈＢＴＣ５０、改変分子Ａおよび改変分子Ｂを添加した場合においても、蛍光染色された細胞、すなわちインスリンを産生するβ細胞に分化した細胞が観察された。
また、インスリン産生細胞の出現頻度は、ｈＢＴＣ８０、ｈＢＴＣ５０、改変分子Ａおよび改変分子Ｂ間で差が見られなかった。
【００８４】
実施例２７
ＰＬＡＰ発現ＡＲ４２Ｊ細胞を用いたβ細胞分化誘導活性の測定
３−１） ヒト胎盤アルカリフォスファターゼ遺伝子発現ベクターの構築
β細胞への分化促進活性はインスリンプロモーターの下流にレポーターとしてアルカリフォスファターゼ遺伝子を連結したものにより形質転換されたＡＲ４２Ｊ細胞を用いても実施した。すなわちベーターセルリンによりβ細胞へと分化した細胞はアルカリフォスファターゼを産生し、このアルカリフォスファターゼの活性を測定することによりβ細胞への分化促進活性を定量的に測定することが可能である。
ラット尾から常法に従ってゲノムＤＮＡを調製した。このＤＮＡをテンプレートとして、既報のラットインスリンＩＩ遺伝子プロモーターの塩基配列（GenBank:Accession No. J00748）をもとに合成したプライマーＲＩ−１：５'−ＡＧＡＧＴＣＡＡＧＧＡＴＣＣＣＣＣＡＡＣＣＡＣＴ−３'およびプライマーＲＩ−３：５'−ＡＧＣＴＧＧＴＣＡＣＴＴＡＧＧＧＣＴＧＧＧＧ−３'を用いてＰＣＲ法によりインスリンプロモーター領域０．７５ Ｋｂを増幅した。さらに、このＰＣＲ産物をテンプレートとしてプライマーＲＩ−１Ｃｌａ：５'−ＧＡＡＴＣＧＡＴＡＧＡＧＴＣＡＡＧＧＡＴＣＣＣＣＣＡ−３'およびプライマーＲＩ−３Ｘｈｏ：５'−ＧＡＣＴＣＧＡＧＣＴＧＧＴＣＡＣＴＴＡＧＧＧ−３'を用いてＰＣＲを行った。増幅された０．７５ Ｋｂ ＤＮＡ断片を単離し、ｐＴ７Ｂｌｕｅベクター（Novagen 69820-1）に組み込んで得られたプラスミドｐＴＢ１８８１を用いてクローニングされた断片の塩基配列を決定し、ラットインスリンＩＩプロモーターであることを確認した。プラスミドｐＴＢ１８８１をＸｈｏ Ｉ−Ｃｌａ Ｉで切断してラットインスリンプロモーターである０．７３ｋｂ ＤＮＡ断片を得た。次にヒト胎盤アルカリフォスファターゼ（ＰＬＡＰ）をコードする２．０ ｋｂ ｃＤＮＡ（J. Bergerら、Gene 66, 1(1988)）の発現プラスミドｐＴＢ１３３０をＸｈｏ Ｉ−Ｈｉｎｄ ＩＩＩで切断して得られた２．７ｋｂ ＤＮＡ断片（ＰＬＡＰ ｃＤＮＡ、ＳＶ４０由来スプライシング部位およびポリＡ付加部位、ｐＢＲ３２２由来ｏｒｉおよびアンピシリン耐性遺伝子を含む）を単離し、前述のラットインシュリンプロモーター領域０．７３ｋｂ Ｘｈｏ Ｉ−Ｃｌａ Ｉ断片をＴ４ ＤＮＡリガーゼ反応によって連結してプラスミドｐＴＢ１８９８を得た。
【００８５】
３−２）ＰＬＡＰ発現ＡＲ４２Ｊ細胞の構築
ＡＲ４２Ｊ細胞にＰＬＡＰ発現プラスミドｐＴＢ１８９８とＴｎ５のｎｅｏ^r遺伝子を含むプラスミドｐＭＣｎｅｏｐｏｌｙ Ａ（STRATAGENE）をトランスフェクション試薬ＴｒａｎｓＩＴ^TM−ＬＴ１（Mirus、PenVera Corporation）を用いて同時に導入した。導入後の細胞は１０％牛胎児血清を加えたＤＭＥＭで２日間培養した後、Ｇ４１８（ゲネチシン、GIBCO BRL）８００μｇ／ｍＬを添加した選択培地にかえて培養を続けた。Ｇ４１８耐性となって生育した細胞を限界希釈法によりクローンを単離した。
各クローンの細胞を２４穴プレートに播き、８０残基型ＢＴＣを２０ｎｇ／ｍＬ添加および無添加で４日間培養し、培養上清を集めて６５℃、３０分間熱処理を行った後、培地中のアルカリフォスファターゼ活性を測定した。８０残基型ベータセルリン添加時にアルカリフォスファターゼ活性の上昇が認められるクローンを選択し、その中の1クローンＡＲ７１−１０４を以下の検討に用いた。
３−３）ＰＬＡＰ発現ＡＲ４２Ｊ細胞を用いたβ細胞分化誘導活性の測定
各種濃度の上記で調製したｈＢＴＣ５０、改変型分子Ａおよび改変型分子Ｂから選ばれる１種と、１０％ウシ胎児血清とを含むダルベッコ改変イーグルＭＥＭ培地に、上記３−２）で構築したＰＬＡＰ発現ＡＲ４２Ｊ細胞を３×１０⁴細胞／ｍＬになるように懸濁し、１００μＬを９６ウェルプレートに播き、炭酸ガスインキュベーター内（５％ 炭酸ガス、９５％空気）において３７℃で５日間培養した。５日後に培養上清をサンプリングし、６５℃で３０分間処理した後、５０μＬをあらかじめ５０μＬの２ＸＳＥＡＰ（２Ｍ ジエタノールアミン、１ｍＭ ＭｇＣｌ₂、２０ｍＭ ホモアルギニン）を添加した９６ウェルマイクロプレートに加えた。３７℃で１０分間保った後、２０ｍｇ／ｍＬのｐ−ニトロフェニルリン酸（シグマ社）を１０μＬ添加し３７℃で１６時間反応を行い、４０５ｎｍの吸光度Ａ₄₀₅を測定した。
ｈＢＴＣ５０添加時の最大吸光度を１００％とした際の５０％の活性を示すのに必要なタンパク濃度（ＥＤ₅₀）を〔表１６〕に示す。
【表１６】

【００８６】
参考例１
５０残基型ヒトベータセルリン（ｈＢＴＣ５０）発現プラスミドｐＴＢ１９７６の構築
８０残基型ｈＢＴＣ ｃＤＮＡを組み込んだプラスミドｐＴＢ１５１６（特開平６−８７８９４号公報記載；受託番号ＦＥＲＭ ＢＰ−３８３６，受託番号ＩＦＯ １５２８２）をテンプレートとして、プライマーＢＴ−９５ｈ：５'−ＡＧＣＡＴＡＴＧＣＧＧＡＡＡＧＧＣＣＡＣＴＴＣＴＣＴＡＧＧＴ−３'およびＢＴ−９４ｈ：５'−ＣＴＧＧＡＴＣＣＴＡＧＴＡＡＡＡＣＡＡＧＴＣＡＡＣＴＣＴＣＴ−３'を用いてＰＣＲを行った。ｈＢＴＣのＣ末５０残基型の５'末端に翻訳開始コドンおよびＮｄｅＩサイトを、３'末端に終止コドンおよびＢａｍＨＩサイトをそれぞれ挿入したＰＣＲ産物をＮｄｅＩおよびＢａｍＨＩで消化し、Ｔ７ファージのΦ１０プロモーターを保持する発現用プラスミドｐＥＴ−３ｃ（Ｎｏｖａｇｅｎ）のＮｄｅＩ−ＢａｍＨＩ部位に ＤＮＡ Ｌｉｇａｔｉｏｎ Ｋｉｔ Ｖｅｒ．２（Ｔａｋａｒａ）を用いて組み込み、ｈＢＴＣ５０の発現プラスミドｐＴＢ１９７６を作製した。組み込んだｃＤＮＡの塩基配列はＡＢＩ社のＤＮＡシーケンサー（ＡＢＩ３７７ ＤＮＡ Ｓｅｑｕｅｎｃｅｒ）により確認した。
プラスミドｐＴＢ１９７６の構築の概略を〔図１０〕に示す。
【００８７】
【発明の効果】
本発明のベータセルリンムテインまたはその塩は、ＢＴＣ活性を保持したまま、ＥＧＦ活性が減弱されており、抗原性の問題もないことから、優れた糖尿病治療薬として有用である。
【００８８】
【配列表】

【００８９】
【図面の簡単な説明】
【図１】７７残基型（Ｃ末端３残基欠失型）ベータセルリン発現プラスミドの構築図を示す。
【図２】７６残基型（Ｃ末端４残基欠失型）ベータセルリン発現プラスミドの構築図を示す。
【図３】実施例７で行われたベータセルリンムテインの電気泳動の結果を示す図を示す。
【図４】実施例８で行われた³Ｈ−チミジンの細胞への取り込み結果を示す図を示す。
【図５】実施例９に記載のβ細胞に分化した細胞に関する蛍光顕微鏡写真図を示す。
【図６】実施例１２で行われたβ細胞分化促進活性の結果を示す図を示す。
【図７】２−７６残基型（Ｎ末端１残基、Ｃ末端４残基欠失型）ベータセルリンの発現プラスミドの構築図を示す。
【図８】２４−７６残基型（Ｎ末端２３残基、Ｃ末端４残基欠失型）ベータセルリンの発現プラスミドの構築図を示す。
【図９】実施例１９で行われた泳動後のゲルの染色結果を示す図を示す。
【図１０】参考例１記載のプラスミドｐＴＢ１９７６の構築の概略を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a mutein (hereinafter referred to as a variant) having reduced proliferation promoting activity (EGF activity) of epithelial cells such as smooth muscle cells while retaining the differentiation promoting activity (BTC activity) of betacellulin (BTC) into pancreatic β cells. Or a modified molecule) and a production method thereof.
[0002]
[Prior art]
Human betacellulin is a protein factor consisting of 80 amino acids excised from a precursor consisting of 178 amino acids, and its entire amino acid sequence has been clarified [Sasada et al .; Biochemical and Biophysical Research Communications (Biochem). Biophys. Res. Commun.), 190: 1173 (1993)]. Although the amount of betacellulin present in the living body is extremely small and difficult to obtain, a production method using a gene recombination technique is described in JP-A-6-87894. Betacellulin was initially found as a factor having a growth promoting activity on mouse 3T3 cells, but later became known to have a growth promoting activity (EGF activity) on vascular smooth muscle cells and retinal pigment epithelial cells. [Shing et al .; Science, 259: 1604 (1993)]. Further, betacellulin has an action (BTC activity) that acts on undifferentiated hepatocytes of the pancreas and promotes differentiation into pancreatic β cells that produce insulin [Mashima et al .; Journal of Clinical Immobilization (J. Clin. Invest.), 97: 1647 (1996)], it is considered useful as a preventive / therapeutic agent for diabetes (eg, insulin-dependent diabetes) and pancreatic dysfunction in diabetes [Miyagawa et al., 1997 Japan Diabetes Society Abstracts, 125].
[0003]
[Problems to be solved by the invention]
However, since betacellulin has the above-mentioned proliferation promoting activity on vascular smooth muscle cells and retinal pigment epithelial cells, these proliferation activities are problematic when applied as a therapeutic agent for diabetes.
Therefore, if it is possible to reduce the proliferation promoting activity against vascular smooth muscle cells, etc. while maintaining the differentiation promoting action to pancreatic β cells, the potential as a pharmaceutical can be increased. Such betacellulin variants are not known.
[0004]
[Means for Solving the Problems]
As a result of various studies on the modified betacellulin, the present inventors can reduce the proliferation promoting activity of smooth muscle cells and the like while maintaining the differentiation promoting activity into pancreatic β cells by modifying the betacellulin. As a result of the first discovery of a modified betacellulin variant that does not cause antigenic problems when administered in vivo and continued research, the present invention was completed. That is, the present invention relates to a modified form of betacellulin and a method for obtaining the modified form.
[0005]
That is, the present invention
(1) Betacellulin mutein or a salt thereof that retains the differentiation promoting activity of pancreatic β cells and has reduced epithelial cell proliferation promoting activity;
(2) Betacellulin mutein or a salt thereof according to (1) above, wherein the ratio of pancreatic β cell differentiation promoting activity to epithelial cell proliferation promoting activity is twice or more that of betacellulin;
(3) 1 to 40 amino acid residues may be deleted from the N-terminus of betacellulin, and in the 1st to 4th amino acid residues from the C-terminus, the third amino acid residue Leu or The betacellulin mutein or salt thereof according to (1) above, wherein 1 to 4 amino acid residues including the 4th amino acid residue Asp from the C-terminal are deleted or substituted with another amino acid residue or another peptide chain ;
(4) The betacellulin mutein or salt thereof according to (3) above, wherein 1 to 40 amino acid residues are deleted from the N-terminus;
(5) (1) amino acid sequence represented by SEQ ID NO: 1, (2) amino acid sequence in which 1 to 40 amino acids have been deleted from the N-terminal of the amino acid sequence represented by SEQ ID NO: 1, (3) The beta according to (3) above, which comprises an amino acid sequence represented by SEQ ID NO: 2 or (4) an amino acid sequence from which 1 to 40 amino acids have been deleted from the N-terminus of the amino acid sequence represented by SEQ ID NO: 2. Serulin mutein or a salt thereof;
(6) (1) amino acid sequence represented by SEQ ID NO: 1, (2) amino acid sequence represented by SEQ ID NO: 2, (3) amino acid sequence represented by SEQ ID NO: 3, or (4) SEQ ID NO: : Betacellulin mutein or a salt thereof according to (3) above, which contains an amino acid sequence represented by 4;
(7) The betacellulin mutein or a salt thereof according to (3), which comprises (1) the amino acid sequence represented by SEQ ID NO: 37 or (2) the amino acid sequence represented by SEQ ID NO: 38;
(8) 1 to 30 amino acid residues at the N-terminus of betacellulin may be deleted, and 1 to 30 amino acid residues between the 22nd and 23rd amino acid residues from the C-terminus The betacellulin mutein or salt thereof according to (1), wherein 5 amino acid residues are inserted;
(9) The betacellulin mutein or salt thereof according to (8) above, wherein 1 to 30 amino acid residues at the N-terminus of betacellulin are deleted;
(10) The betacellulin mutein or a salt thereof according to (8), which has the amino acid sequence represented by SEQ ID NO: 44;
(11) The betacellulin mutein or a salt thereof according to (8), which has the amino acid sequence represented by SEQ ID NO: 45;
(12) The transformant transformed with the recombinant vector containing DNA encoding the betacellulin mutein according to (1) is cultured to produce the betacellulin mutein (1) A process for producing the described betacellulin mutein or a salt thereof;
(13) A pharmaceutical composition comprising the betacellulin mutein or a salt thereof according to (1);
(14) The pharmaceutical composition according to (9) above, wherein the composition is a diabetes preventive / therapeutic agent.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
As described above, the modified betacellulin of the present invention (betacellulin mutein) has reduced or attenuated EGF activity while maintaining BTC activity.
Here, betacellulin means Asp Gly Asn Ser Thr Arg as described in Sasada et al .; Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 190: 1173 (1993). Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Leu Phe Tyr (SEQ ID NO: 35) Means a polypeptide containing the amino acid sequence represented by (SEQ ID NO: 35).
Examples of betacellulin muteins that retain BTC activity include betacellulin muteins having an activity of 20% or more, preferably 50% or more of the BTC activity of betacellulin.
Examples of betacellulin muteins with reduced EGF activity include betacellulin muteins having an activity of about 20% or less, preferably 15% or less of the EGF activity of betacellulin.
[0007]
In the betacellulin mutein of the present invention in which the EGF activity is reduced while maintaining the BTC activity, the ratio of the differentiation promoting activity of pancreatic β cells to the epithelial cell proliferation promoting activity is more than twice that of betacellulin. Betacellulin mutein or a salt thereof is preferred.
Specifically, the betacellulin mutein of the present invention with reduced EGF activity while retaining BTC activity may specifically have 1 to 40 amino acid residues deleted from the N-terminus of betacellulin, 1 to 4 amino acid residues from 1 to 4 amino acid residues including the third amino acid residue Leu from the C terminus or the fourth amino acid residue Asp from the C terminus are deleted or other amino acid residues Β-cellulin mutein substituted with a group or other peptide chain, or a salt thereof, and more specifically, the aforementioned beta in which 1 to 40 or 1 to 20 amino acid residues are deleted from the N-terminus Cellulin mutein.
[0008]
Other “substitution with other amino acid residues or other peptide chains” in the above-mentioned “betacellulin mutein substituted with other amino acid residues or other peptide chains” means that EGF is maintained while retaining BTC activity. The amino acid residue or the type of peptide chain is not particularly limited as long as the substitution does not impair the characteristic that EGF is attenuated while retaining the BTC activity of the betacellulin mutein of the present invention with reduced activity. Absent. Specific examples of the above “other amino acids” include nonpolar (hydrophobic) amino acids (eg, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, etc.), polar (neutral) amino acids (eg, glycine) , Serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc.) positively charged (basic) amino acids (eg, arginine, lysine, histidine), negatively charged (acidic) amino acids (eg, aspartic acid, glutamic acid) Etc.). As a specific example of the above “other peptide chain”, it means a peptide chain formed by combining two or more of the above “other amino acids”, preferably a peptide chain composed of 2 to 4 amino acid residues. Etc.
[0009]
“1 to 40 amino acid residues may be deleted from the N-terminus of betacellulin, and in the 1st to 4th amino acid residues from the C-terminus, the third amino acid residue Leu from the C-terminus or from the C-terminus Specific examples of “betacellulin mutein in which 1 to 4 amino acid residues including the fourth amino acid residue Asp are deleted or substituted with other amino acid residues or other peptide chains”
(1) Amino acid sequence from the N-terminal to the 76th position of betacellulin (Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val) In the first to fourth amino acid residues (Asp Leu Phe Tyr), 1 to 4 amino acid residues including the third amino acid residue Leu from the C terminus or the fourth amino acid residue Asp from the C terminus are deleted. Or betacellulin muteins substituted with other amino acid residues or other peptide chains, for example,
▲ 1 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val (SEQ ID NO: 2)
▲ 2 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp (SEQ ID NO: 1)
▲ 3 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Leu Phe Tyr (SEQ ID NO: 5) ,
▲ 4 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Leu Phe (SEQ ID NO: 6)
▲ 5 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Leu (SEQ ID NO: 7)
▲ 6 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Phe Tyr (SEQ ID NO: 8) ,
▲ 7 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Phe (SEQ ID NO: 9) and
(8) Betacellulin mutein in which the third amino acid residue (Leu) from the C-terminal of betacellulin is substituted with another amino acid residue.
Among these, a betacellulin mutein containing the amino acid sequence represented by SEQ ID NO: 1 or a betacellulin mutein represented by SEQ ID NO: 2 is particularly preferable.
[0010]
(2) Amino acid residues 1 to 40 from the N-terminal of betacellulin (Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys)
The 41st to 76th amino acid sequence from the N-terminus (Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val) 1 to 4 amino acid residues (Asp Leu Phe Tyr) from the C terminus, including the third amino acid residue Leu from the C terminus or the fourth amino acid residue Asp from the C terminus Betacellulin muteins in which amino acid residues have been deleted or replaced with other amino acid residues or other peptide chains, preferably
(I) amino acid residues 1 to 37 from the N-terminal of betacellulin (Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg)
38th to 76th amino acid sequence from the N-terminus (Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg 1 to 4 including the third amino acid residue Leu from the C terminus or the fourth amino acid residue Asp from the C terminus in the first to fourth amino acid residues (Asp Leu Phe Tyr) from the C terminus. Betacellulin mutein in which 4 amino acid residues are deleted or replaced by other amino acid residues or other peptide chains,
(Ii) the first amino acid residue (Asp) from the N-terminus of betacellulin may be deleted;
N-terminal 2nd to 76th amino acid sequence (Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val) In the amino acid residue (Asp Leu Phe Tyr), 1 to 4 amino acid residues including the third amino acid residue Leu from the C terminus or the fourth amino acid residue Asp from the C terminus are deleted or other amino acid residues Betacellulin muteins substituted with groups or other peptide chains,
(Iii) The 1st to 23rd amino acid residues from the N-terminus of betacellulin (Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala) may be deleted ,
24th to 76th amino acid sequence from the N-terminus (Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val) In the first to fourth amino acid residues (Asp Leu Phe Tyr) from the C terminus, the third amino acid residue Leu or Examples include betacellulin muteins in which 1 to 4 amino acid residues including the 4th amino acid residue Asp from the C-terminal are deleted or substituted with other amino acid residues or other peptide chains.
[0011]
(a) Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val ( Betacellulin mutein containing the amino acid sequence represented by SEQ ID NO: 4),
(b) Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Betacellulin mutein containing the amino acid sequence represented by (SEQ ID NO: 3),
(c) Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Leu Betacellulin mutein containing the amino acid sequence represented by Phe Tyr (SEQ ID NO: 10),
(d) Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Leu Betacellulin mutein containing the amino acid sequence represented by Phe (SEQ ID NO: 11),
(e) Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Leu Betacellulin mutein containing the amino acid sequence represented by (SEQ ID NO: 12),
(f) Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Betacellulin mutein containing the amino acid sequence represented by Phe Tyr (SEQ ID NO: 13),
(g) Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Betacellulin mutein containing the amino acid sequence represented by Phe (SEQ ID NO: 14),
(h) a betacellulin mutein in which the third amino acid residue (Leu) from the C-terminus is substituted with another amino acid residue in the partial peptide represented by the amino acid sequence 31 to 80 from the N-terminus of betacellulin;
(i) Gly Asn Ser Thr Arg Ser Pto Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val (SEQ ID NO: 37)
(j) Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Examples thereof include betacellulin mutein containing the amino acid sequence represented by Ala Arg Cys Glu Arg Val (SEQ ID NO: 38).
[0012]
Among them, betacellulin mutein containing the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 37 or SEQ ID NO: 38 is preferable, and more preferably SEQ ID NO: 37 or SEQ ID NO: 38. It is a betacellulin mutein containing the amino acid sequence represented by these. Most preferred is a betacellulin mutein containing the amino acid sequence represented by SEQ ID NO: 38.
Among the preferred examples of the betacellulin mutein of the present invention described in (1) and (2) above, particularly preferred are (1) the amino acid sequence represented by SEQ ID NO: 1, and (2) SEQ ID NO: 1. An amino acid sequence in which 1 to 40 amino acids are deleted from the N-terminus of the amino acid sequence represented, (3) an amino acid sequence represented by SEQ ID NO: 2, and (4) an amino acid sequence represented by SEQ ID NO: 2. An amino acid sequence in which 1 to 40 amino acids have been deleted from the N-terminal, (5) an amino acid sequence represented by SEQ ID NO: 37, or (6) a betacellulin mutein comprising an amino acid sequence represented by SEQ ID NO: 38 is there.
More preferably, (1) the amino acid sequence represented by SEQ ID NO: 1, (2) the amino acid sequence represented by SEQ ID NO: 2, (3) the amino acid sequence represented by SEQ ID NO: 3, (4) sequence No. 4 amino acid sequence, (5) amino acid sequence represented by SEQ ID NO: 37, or (6) betacellulin mutein containing the amino acid sequence represented by SEQ ID NO: 38. Particularly preferred is a betacellulin mutein containing the amino acid sequence represented by SEQ ID NO: 38.
[0013]
Specifically, the betacellulin mutein of the present invention in which EGF activity is reduced while retaining BTC activity, (1) 1 to 30 amino acid residues at the N-terminus of betacellulin may be deleted. , Betacellulin mutein or a salt thereof in which 1 to 5 amino acid residues are inserted between the 22nd amino acid residue and the 23rd amino acid residue from the C-terminus, (2) SEQ ID NO: 45 Examples also include betacellulin mutein having the amino acid sequence represented or a salt thereof.
Specific examples of the “amino acid” of the above-mentioned “betacellulin mutein (modified molecule) with an inserted amino acid residue” include nonpolar (hydrophobic) amino acids (eg, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan) ), Polar (neutral) amino acids (eg, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc.), positively charged (basic) amino acids (eg, arginine, lysine, histidine, etc.), Examples include negatively charged (acidic) amino acids (eg, aspartic acid, glutamic acid, etc.). Asparagine, proline and serine are preferred. The “peptide chain” refers to a peptide chain formed by bonding 2 to 5 “amino acids”, preferably a peptide chain composed of 2 to 4 amino acid residues.
[0014]
Examples of “betacellulin from which 1 to 30 amino acid residues at the N-terminus may be deleted” include (1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 35, (2) N A betacellulin-modified molecule in which 1 to 30 amino acid residues at the terminal have been deleted; (3) an amino acid residue (for example, 1 to 5 amino acids) in the 1st to 30th amino acid sequences at the N-terminus of betacellulin; Betacellulin-modified molecules into which amino acid residues are inserted).
The “betacellulin-modified molecule lacking 1 to 30 amino acid residues at the N-terminal” includes (1) a molecule lacking 1 to 30 amino acid residues on the N-terminal side of betacellulin; 2) Examples include those in which 1 to 30 amino acid residues on the N-terminal side of betacellulin are deleted and amino acid residues (eg, 1 to 5 amino acid residues) are added.
[0015]
“1 to 30 amino acid residues at the N-terminal of betacellulin may be deleted, and the 22nd amino acid residue from the C-terminal (that is, the N-terminal of the amino acid sequence represented by SEQ ID NO: 35) To the 58th amino acid residue: Gln) and the 23rd amino acid residue (that is, the 59th amino acid residue: Thr from the N-terminal of the amino acid sequence represented by SEQ ID NO: 35) “A betacellulin-modified molecule having 1 to 5 amino acid residues inserted” means that any part of the 1 to 30 amino acid residues at the N-terminal of betacellulin may be deleted. It may be a betacellulin modified molecule in which 1 to 5 amino acid residues are inserted between the 22nd amino acid residue and the 23rd amino acid residue.
▲ 1 ▼ Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Asn Pro Ser Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Leu Phe Tyr (SEQ ID NO: 46) A betacellulin-modified molecule,
(2) 1 to 30 amino acid residues from the N-terminus of betacellulin (Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys) Is missing and
A betacellulin-modified molecule in which 1 to 5 amino acid residues are inserted between the 22nd amino acid residue and the 23rd amino acid residue from the C-terminal, for example, Arg Lys Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly Arg Cys Arg Phe Val Val Ala Glu Gln Asn Pro Ser Thr Pro Ser Cys Val Cys Asp Glu Gly Tyr Ile Gly Ala Arg Cys Glu Arg Val Asp Leu Phe Tyr (SEQ ID NO: 44) And a betacellulin-modified molecule containing the amino acid sequence represented by
The “betacellulin-modified molecule” of the present invention is preferably a betacellulin-modified molecule containing the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45.
[0016]
In the present specification, betacellulin mutein has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. In addition, these betacellulin muteins are usually carboxyl groups (—COOH) or carboxylates (—COO) at the C-terminus. ^- ) But the C-terminus is an amide (-CONH ₂ ) Or ester (—COOR). Examples of R in the ester include C such as methyl, ethyl, n-propyl, isopropyl or n-butyl. _1-6 C such as alkyl group, cyclopentyl, cyclohexyl, etc. _3-8 C such as cycloalkyl group, phenyl, α-naphthyl, etc. _6-12 Phenyl-C such as aryl group, benzyl, phenethyl, benzhydryl _1-2 Alkyl or α-naphthyl-C such as α-naphthylmethyl _1-2 C such as alkyl _7-14 Examples thereof include an aralkyl group and a pivaloyloxymethyl group.
Further, the betacellulin muteins of the present invention include those in which Met is added to the N-terminus.
[0017]
As the salt of betacellulin mutein of the present invention, a salt with a physiologically acceptable base (such as an alkali metal) or an acid (organic acid, inorganic acid) is used. Salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, Salts with tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
The betacellulin mutein of the present invention can be obtained by, for example, treating a betacellulin obtained by genetic engineering or beta-tumor cell culture described in JP-A-6-87894 with a known protease treatment, preferably a carboxypeptidase, more preferably bovine pancreas It can also be prepared by treating with carboxypeptidase A. Moreover, it can also manufacture according to the below-mentioned peptide synthesis method. It can also be produced by culturing a transformant containing DNA encoding betacellulin mutein, which will be described later.
When betacellulin is produced from tissues or cells of humans or warm-blooded animals, the tissues or cells of humans or warm-blooded animals are homogenized and then extracted with acid, etc., and the extract is subjected to salting out, dialysis, gel filtration , And can be purified and isolated by combining chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like.
[0018]
As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, to produce the desired betacellulin mutein by condensing the partial peptide or amino acid that can constitute the betacellulin mutein of the present invention and the remaining part, and removing the protecting group when the product has a protecting group Can do. Examples of known condensation methods and protecting group removal include the methods described in (1) to (5) below.
▲ 1 ▼ M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966) ▲ 2 ▼ Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
▲ 5 ▼ Supervision by Yajima Haruaki, Development of follow-up medicines Volume 14 Peptide synthesis Hirokawa Shoten
Further, after the reaction, the polypeptide of the present invention can be purified and isolated by a combination of usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the polypeptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted to a free form by a known method. Can do.
[0019]
As the amide form of betacellulin mutein, a commercially available resin for peptide synthesis suitable for amide formation can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenyl. Examples include acetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. . Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target peptide according to various condensation methods known per se. At the end of the reaction, the peptide is cut out from the resin and at the same time, various protecting groups are removed, and if necessary, intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the desired amide form.
For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide and the like. For activation by these, a protected amino acid together with a racemization inhibitor (eg, HOBt, HOBt, etc.) is added directly to the resin, or a pre-protected amino acid such as a symmetric anhydride or HOBt ester or HOBt ester is added. It can be added to the resin after activation. The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents that are known to be usable for the peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and sulfoxides such as dimethyl sulfoxide. , Tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from a range that is known to be usable for peptide bond formation reaction, and is usually selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.
[0020]
Examples of the protecting group for the amino group of the raw material amino acid include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoro Acetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like can be mentioned. Examples of the protecting group for the carboxyl group include C described above as R. _1-6 Alkyl group, C _3-8 A cycloalkyl group, C _7-14 In addition to the aralkyl group, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl group and benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, trityl hydrazide and the like can be mentioned.
The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, groups derived from carbon such as benzyloxycarbonyl groups and ethoxycarbonyl groups. Examples of groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a tertiary butyl group.
As a protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like.
[0021]
Examples of the activated carboxyl group of the raw material include the corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyano Methyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOBt)] and the like. Examples of the activated amino group of the raw material include the corresponding phosphoric acid amide.
Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid. And acid treatment with trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like. The elimination reaction by the acid treatment is generally performed at a temperature of −20 ° C. to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, 1 It is effective to add a cation scavenger such as 1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.
The method for introducing a protecting group into a functional group that should not participate in the reaction, the method for removing the protecting group, and the method for activating the functional group involved in the reaction may be carried out according to a method known per se or a method analogous thereto. .
[0022]
As another method for obtaining an amide form of betacellulin mutein, first, the α-carboxyl group of the carboxyl terminal amino acid is amidated, then the peptide chain is extended to the desired chain length on the amino group side, and then the peptide A peptide in which only the protecting group of the α-amino group at the N-terminus of the chain is removed and a peptide (or amino acid) in which only the protecting group of the carboxyl group at the C-terminus is removed are prepared, and both peptides are mixed as described above. Condense in. The details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above-described method to obtain the desired crude polypeptide. This crude polypeptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide of the desired polypeptide.
To obtain an ester of betacellulin mutein, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired polypeptide is obtained in the same manner as the amide of the polypeptide. be able to.
[0023]
As DNA encoding the betacellulin mutein of the present invention, (1) 1 to 40 amino acid residues from the N-terminus of betacellulin may be deleted, and the 1st to 4th amino acid residues from the C-terminus 1 to 4 amino acid residues including the third amino acid residue Leu from the C terminus or the fourth amino acid residue Asp from the C terminus are deleted or substituted with other amino acid residues or other peptide chains. (2) 1 to 30 amino acid residues at the N-terminus of betacellulin may be deleted, and the 22nd and 23rd amino acid residues from the C-terminus A betacellulin mutein having 1 to 5 amino acid residues inserted in between, (3) a betacellulin mutein having the amino acid sequence represented by SEQ ID NO: 45 If DNA containing a DNA may be any. Specifically, beta containing the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 14, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 of the present invention. Examples thereof include those containing a base sequence encoding cellulin mutein.
More specifically, (1) DNA encoding betacellulin mutein containing the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 14 is represented by SEQ ID NO: 15 to SEQ ID NO: 28. DNA containing DNA having a base sequence, and (2) DNA encoding betacellulin mutein containing the amino acid sequences represented by SEQ ID NO: 37 and SEQ ID NO: 38 are represented by SEQ ID NO: 42 and SEQ ID NO: 43. (3) DNA containing a DNA having the base sequence represented by SEQ ID NO: 47 to SEQ ID NO: 49, (4) DNA containing the DNA having the base sequence shown in (4) above under stringent conditions (1) ) To (3) DNA derived from a mammal that hybridizes with the sequence defined in (3), (5) No (1) above due to degeneracy of the genetic code (4) no sequence hybridize which are defined, but a DNA encoding a polypeptide having the same amino acid sequence is used. Hybridization can be performed according to a method known per se or a method analogous thereto. Examples of the stringent conditions include 42 ° C., 50% formamide, 4 × SSPE (1 × SSPE = 150 mM NaCl, 10 mM NaH). ₂ PO _Four ・ H ₂ O, 1 mM EDTA pH 7.4), 5 × Denhart solution, 0.1% SDS.
[0024]
The expression vector for betacellulin mutein of the present invention used when the betacellulin mutein of the present invention is produced by culturing a transformant containing DNA encoding betacellulin mutein is, for example, (1) the present invention The desired DNA fragment can be excised from the DNA encoding betacellulin mutein, and (2) the DNA fragment can be ligated downstream of the promoter in an appropriate expression vector.
Examples of the vector include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage, and the like. Animal viruses such as bacteriophage, retrovirus, vaccinia virus and baculovirus are used.
The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.
When the host for transformation is an animal cell, SV40-derived promoters, retrovirus promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters, SRα promoters and the like can be used. When the host is Escherichia, trp promoter, T7 promoter, lac promoter, recA promoter, λP _L When the host is Bacillus, the promoter, lpp promoter, etc. are SPO1, SPO2, promoter, etc. When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter, etc. Is preferred. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.
[0025]
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selection markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter Amp). ^r And neomycin resistance gene (hereinafter sometimes abbreviated as Neo, G418 resistance) and the like. In particular, CHO (dhfr ^- ) When using the DHFR gene as a selection marker using cells, it can also be selected by a medium not containing thymidine.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the betacellulin mutein. When the host is Escherichia, phoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc. are used in yeast. In some cases, mating factor α (MFα) signal sequence, invertase signal sequence, etc. When the host is an animal cell, for example, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. Can be used respectively.
A transformant can be produced using the vector containing the DNA encoding the betacellulin mutein thus constructed.
[0026]
As the host, for example, Escherichia, Bacillus, yeast, insect or insect cells, animal cells, and the like are used.
Examples of the genus Escherichia include Escherichia coli K12 / DH1 [Proc. Natl. Acad. Sci. USA], 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], MM294 [Procedure] Of the National Academy of Sciences of The USA (Proc. Natl. Acad. Sci. USA), 73 volumes, and 4174 (1976)] is used.
Examples of the genus Bacillus include Bacillus subtilis MI114 [Gen, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984). ] Is used.
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R. ^- NA87-11A, DKD-5D, 20B-12, etc. are used.
Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].
Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five derived from eggs of Trichoplusia ni ^TM Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, silkworm-derived cell lines (Bombyx mori N; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells [Vaughn, JL et al., In Vitro, Vol. 13, 213-217 (1977)].
[0027]
Examples of animal cells include monkey COS-7 cells, Vero cells, Chinese hamster cells CHO, DHFR gene-deficient Chinese hamster cells CHO (dhfr). ^- CHO cells), mouse L cells, mouse 3T3 cells, mouse myeloma cells, human HEK293 cells, human FL cells, 293 cells, C127 cells, BALB3T3 cells, Sp-2 / O cells and the like.
In order to transform Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Proceedings of the National Academy of Sciences of the USA ), Gene, Vol. 17, 107 (1982).
In order to transform Bacillus genus bacteria, for example, the method described in Molecular & General Genetics, 168, 111 (1979), etc. is performed.
For transforming yeast, see, for example, Proc. Natl. Acad. Sci. USA, 75, 1929 (1978), Proc. Of the National Academy of Sciences of the USA. This is done according to the method described.
Insect cells or insects are transformed according to the method described in, for example, Bio / Technology, Vol. 6, pp. 47-55 (1988).
Animal cells are transformed according to the method described in, for example, Virology, Vol. 52, 456 (1973).
[0028]
Examples of methods for introducing an expression vector into cells include the lipofection method [Felgner, PL et al. Proceedings of the National Academy of Sciences of the National Academy of Sciences of the United States of America), 84, 7413 (1987)], calcium phosphate method [Graham, FL and van der Eb, AJ Virology, 52, 456-467 (1973)], Electroporation (Nuemann, E. et al. Embo Journal (EMBO J.), Vol. 1, pp. 841-845 (1982)).
Thus, a transformant transformed with an expression vector containing DNA encoding the betacellulin mutein of the present invention is obtained.
In addition, as a method of stably expressing the betacellulin mutein of the present invention using animal cells, there is a method of selecting a cell in which the expression vector introduced into the animal cell is incorporated into a chromosome by clone selection. Specifically, a transformant is selected using the above selection marker as an index. Furthermore, stable animal cell lines having high expression ability such as the betacellulin mutein of the present invention can be obtained by repeatedly selecting clones for the animal cells obtained using the selection marker in this way. In addition, when the dhfr gene is used as a selection marker, the MTX concentration is gradually increased and cultured, and a resistant strain is selected to amplify DNA encoding the betacellulin mutein of the present invention together with the dhfr gene in the cell. Thus, animal cell lines with higher expression can be obtained.
[0029]
Producing the betacellulin mutein of the present invention by culturing the above transformant under conditions capable of expressing the DNA encoding the betacellulin mutein of the present invention, and generating and accumulating the betacellulin mutein of the present invention. Can do.
When cultivating a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Or as an organic substance and an inorganic substance, calcium chloride, sodium dihydrogen phosphate, magnesium chloride etc. are mentioned, for example. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972]. Here, in order to make the promoter work efficiently if necessary, a drug such as 3β-indolylacrylic acid can be added.
When the host is Escherichia, the culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
When the host is Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
[0030]
When culturing a transformant whose host is yeast, examples of the medium include a Burkholder minimal medium [Bostian, KL et al., “Procedures of the National Academy of Sciences of Sciences. The USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)] The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an insect cell, an appropriate medium such as Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962), 10% bovine serum) The pH of the medium is preferably adjusted to about 6.2 to 6.4 The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary. .
When cultivating a transformant whose host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium [ Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Journal of the American Medical Association Vol. Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. The pH is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.
Especially CHO (dhfr ^- ) When using cells and the dhfr gene as selectable markers, it is preferable to use a DMEM medium containing dialyzed fetal bovine serum that contains little thymidine.
[0031]
Separation and purification of the betacellulin mutein of the present invention from the culture can be performed, for example, by the following method.
When the betacellulin mutein of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freezing. A method of obtaining a crude polypeptide extract by centrifugation or filtration after disrupting cells or cells by thawing or the like can be used as appropriate. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark, hereinafter may be abbreviated as TM).
When betacellulin mutein is secreted into the culture solution, after completion of the culture, the cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected.
Purification of the polypeptide of the present invention contained in the thus obtained culture supernatant or extract can be performed by appropriately combining per se known separation and purification methods. These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point such as a method, isoelectric focusing method or chromatofocusing is used.
[0032]
When the betacellulin mutein of the present invention thus obtained is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely when it is obtained as a salt, it is known per se. It can be converted into a free form or other salt by a method or a method analogous thereto.
In addition, the betacellulin mutein of the present invention produced by the recombinant may be arbitrarily modified or partially removed by allowing an appropriate protein modifying enzyme to act before or after purification. it can. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The obtained betacellulin mutein may be subjected to a refolding step described in, for example, JP-A-10-191989. Alternatively, N-terminal Met can also be removed by subjecting beta-cellulin mutein with Met added to the N-terminal to the N-terminal Met removal reaction described in JP-A-10-191989.
The presence of the betacellulin mutein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody.
[0033]
The DNA encoding the betacellulin mutein of the present invention or the betacellulin mutein of the present invention or a salt thereof is used for diabetes (for example, insulin-dependent diabetes mellitus (type I diabetes), etc.), pancreatic dysfunction in diabetes, senile insulin secretion decrease It can be used for the development of pharmaceuticals for improving agents such as pancreatic hypofunction associated with cancer and undifferentiated pancreatic cancer (especially prophylactic / therapeutic agents for diabetes (eg, insulin-dependent diabetes)).
Furthermore, the betacellulin mutein of the present invention or a salt thereof, or a DNA encoding the same is useful as a safe and low-toxic pharmaceutical because EGF activity is attenuated and there is no antigenic problem. The betacellulin mutein of the present invention or a salt thereof or a DNA encoding the same is used as an agent for improving diabetes (eg, insulin-dependent diabetes), pancreatic dysfunction in diabetes, pancreatic dysfunction associated with senile decrease in insulin secretion, and the like. It can be used as a therapeutic / preventive agent for diseases such as undifferentiated pancreatic cancer.
[0034]
When the betacellulin mutein of the present invention or a salt thereof or a DNA encoding the same is used as the above-mentioned pharmaceutical, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules or the like with sugar coating or enteric coating as necessary, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of an injection such as a suspension. For example, mixing the compound or salt thereof with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc. in the unit dosage form required for the generally accepted formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection may be formulated according to conventional pharmaceutical manufacturing methods such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. it can.
Examples of aqueous solutions for injection include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, and appropriate solubilizing agents. For example, you may use together with alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), a nonionic surfactant (for example, polysorbate 80 (TM), HCO-50), etc. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent.
Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule.
[0035]
Since the preparation thus obtained is safe and has low toxicity, it is suitable for humans and mammals (for example, mouse, rat, guinea pig, rabbit, sheep, pig, cow, cat, dog, monkey, baboon, chimpanzee, etc.). Can be administered.
The dose of betacellulin mutein or a salt thereof of the present invention varies depending on symptoms, but in the case of oral administration, it is generally from about 0.1 per day in adult diabetic patients (with a body weight of 60 kg). 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but for example, as an injection, for adult diabetic patients (weight 60 kg) It is convenient to administer about 0.01 to 30 mg per dose, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.
[0036]
In the present specification and drawings, when bases, amino acids, and the like are indicated by abbreviations, they are based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: Deoxyribonucleic acid
cDNA: complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: cytosine
EDTA: Ethylenediaminetetraacetic acid
APMSF: (p-amidinophenyl) methanesulfonyl fluoride hydrochloride
SDS: sodium dodecyl sulfate
TFA: trifluoroacetic acid
Gly: Glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: Cysteine
Met: Methionine
Glu: Glutamic acid
Asp: Aspartic acid
Lys: Lysine
Arg: Arginine
His: Histidine
Phe: Phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
NMP: N-methylpyrrolidone
[0037]
In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Me: methyl group
Et: ethyl group
Bu: butyl group
Ph: phenyl group
TC: thiazolidine-4 (R) -carboxamide group
Bom: benzyloxymethyl
PAM: Phenylacetamidomethyl
Tos: p-toluenesulfonyl
HONB: N-hydroxy-5-norbornene-2,3-dicarboximide
Bzl: benzyl group
Z: benzyloxycarbonyl group
Br-Z: 2-bromobenzyloxycarbonyl group
Cl-Z: 2-chlorobenzyloxycarbonyl group
Boc: t-butyloxycarbonyl group
HOBt: 1-hydroxybenztriazole
DCC: N, N′-dicyclohexylcarbodiimide
TFA: trifluoroacetic acid
Fmoc: N-9-fluorenylmethoxycarbonyl group
DNP: dinitrophenyl group
Bum: Tertiary butoxymethyl group
Trt: Trityl group
[0038]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
1 shows the amino acid sequence of betacellulin mutein (BTC1-77) of the present invention.
[SEQ ID NO: 2]
1 shows the amino acid sequence of betacellulin mutein (BTC1-76) of the present invention.
[SEQ ID NO: 3]
3 shows the amino acid sequence of betacellulin mutein (BTC31-77) of the present invention.
[SEQ ID NO: 4]
1 shows the amino acid sequence of betacellulin mutein (BTC31-76) of the present invention.
[SEQ ID NO: 5]
The amino acid sequence of the betacellulin mutein (BTC1-76, 78-80) of this invention is shown.
[SEQ ID NO: 6]
The amino acid sequence of the betacellulin mutein (BTC1-76, 78, 79) of this invention is shown.
[SEQ ID NO: 7]
The amino acid sequence of the betacellulin mutein (BTC1-76, 78) of this invention is shown.
[SEQ ID NO: 8]
The amino acid sequence of the betacellulin mutein (BTC1-77, 79, 80) of this invention is shown.
[SEQ ID NO: 9]
The amino acid sequence of the betacellulin mutein (BTC1-77, 80) of the present invention is shown.
[SEQ ID NO: 10]
The amino acid sequence of the betacellulin mutein (BTC31-76, 78-80) of this invention is shown.
[0039]
[SEQ ID NO: 11]
The amino acid sequence of the betacellulin mutein (BTC31-76, 78, 79) of this invention is shown.
[SEQ ID NO: 12]
The amino acid sequence of the betacellulin mutein (BTC31-76, 78) of the present invention is shown.
[SEQ ID NO: 13]
The amino acid sequence of the betacellulin mutein (BTC31-77, 79, 80) of this invention is shown.
[SEQ ID NO: 14]
The amino acid sequence of the betacellulin mutein (BTC31-77, 79) of this invention is shown.
[SEQ ID NO: 15]
1 shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 1.
[SEQ ID NO: 16]
This shows the base sequence of cDNA encoding the betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 2.
[SEQ ID NO: 17]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 3.
[SEQ ID NO: 18]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 4.
[SEQ ID NO: 19]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 5.
[SEQ ID NO: 20]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 6.
[0040]
[SEQ ID NO: 21]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 7.
[SEQ ID NO: 22]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 8.
[SEQ ID NO: 23]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 9.
[SEQ ID NO: 24]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 10.
[SEQ ID NO: 25]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 11.
[SEQ ID NO: 26]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 12.
[SEQ ID NO: 27]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 13.
[SEQ ID NO: 28]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 14.
[SEQ ID NO: 29]
The base sequence of primer 1 used in Examples 1 and 4 described later is shown.
[SEQ ID NO: 30]
The base sequence of primer 2 used in Examples 1 and 4 described later is shown.
[0041]
[SEQ ID NO: 31]
The base sequence of primer RI-1 used in Example 10 and Example 27 described later is shown.
[SEQ ID NO: 32]
The base sequence of primer RI-3 used in Example 10 and Example 27 described later is shown.
[SEQ ID NO: 33]
The base sequence of primer RI-1Cla used in Example 10 and Example 27 described later is shown.
[SEQ ID NO: 34]
The base sequence of primer RI-3Xho used in Example 10 and Example 27 described later is shown.
[SEQ ID NO: 35]
The amino acid sequence of betacellulin is shown.
[SEQ ID NO: 36]
The base sequence of cDNA encoding betacellulin is shown.
[SEQ ID NO: 37]
1 shows the amino acid sequence of betacellulin mutein (BTC2-76) of the present invention.
[SEQ ID NO: 38]
1 shows the amino acid sequence of betacellulin mutein (BTC24-76) of the present invention.
[SEQ ID NO: 39]
The base sequence of primer 3 used in Example 13 described later is shown.
[SEQ ID NO: 40]
The base sequence of primer 4 used in Examples 13 and 16 described later is shown.
[0042]
[SEQ ID NO: 41]
The base sequence of primer 5 used in Example 16 described later is shown.
[SEQ ID NO: 42]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 37.
[SEQ ID NO: 43]
This shows the base sequence of cDNA encoding betacellulin mutein represented by the amino acid sequence represented by SEQ ID NO: 38.
[SEQ ID NO: 44]
The amino acid sequence of the betacellulin modified molecule (BTC31-58, Asn, Pro, Ser, 59-80) of the present invention is shown.
[SEQ ID NO: 45]
The amino acid sequence of the betacellulin modification molecule | numerator (Asn, Ser, Asp, Ser, Glu, BTC38-80) of this invention is shown.
[SEQ ID NO: 46]
The amino acid sequence of the betacellulin modification molecule | numerator (BTC1-58, Asn, Pro, Ser, 59-80) of this invention is shown.
[SEQ ID NO: 47]
This shows the base sequence of cDNA encoding the betacellulin-modified molecule represented by the amino acid sequence shown in SEQ ID NO: 46.
[SEQ ID NO: 48]
This shows the base sequence of cDNA encoding the betacellulin-modified molecule represented by the amino acid sequence shown in SEQ ID NO: 44.
[SEQ ID NO: 49]
This shows the base sequence of cDNA encoding the betacellulin-modified molecule represented by the amino acid sequence shown in SEQ ID NO: 45.
[SEQ ID NO: 50]
The base sequence of primer BT-95h used in Reference Example 1 and Example 22 described later is shown.
[SEQ ID NO: 51]
The base sequence of primer BT-94h used in Reference Example 1 and Example 23 described later is shown.
[SEQ ID NO: 52]
The base sequence of primer PET-1 used in Example 22 described later is shown.
[SEQ ID NO: 53]
The base sequence of primer BTC-1 used in Example 22 described later is shown.
[SEQ ID NO: 54]
The base sequence of primer BTC-2 used in Example 22 described later is shown.
[SEQ ID NO: 55]
The base sequence of primer BTC-3 used in Example 22 described later is shown.
[SEQ ID NO: 56]
The base sequence of primer BTC-7 used in Example 23 described later is shown.
[0043]
Transformant Escherichia coli [Escherichia coli] MM294 (DE3) / pTCIIBTC77 obtained in Example 1 to be described later has the accession number FERM BP-6684 as of November 24, 1998, METI Biotechnology Industry. Deposited at the Technical Research Institute (NIBH). In addition, it was deposited with the Fermentation Research Institute (IFO) under the accession number IFO 16214 on November 2, 1998.
Transformant Escherichia coli [Escherichia coli] MM294 (DE3) / pTCIIBTC76 obtained in Example 4 to be described later has the accession number FERM BP-6583 dated November 24, 1998, METI Biotechnology Industry. Deposited at the Technical Research Institute. In addition, it was deposited with the Fermentation Research Institute (IFO) under the accession number IFO 16213 on November 2, 1998.
[0044]
Transformant Escherichia coli [Escherichia coli] MM294 (DE3) / pTCIIBTC2-76 obtained in Example 13, which will be described later, has the accession number FERM BP-6948, dated November 24, 1999, by the Ministry of International Trade and Industry. Deposited at the Institute of Engineering and Industrial Technology. It was deposited with the Fermentation Research Institute (IFO) as deposit number IFO 16334 on November 9, 1999.
Transformant Escherichia coli [Escherichia coli] MM294 (DE3) / pTCIIBTC24-76 obtained in Example 16 to be described later has the accession number FERM BP-6949 as of November 24, 1999. Deposited at the Institute of Engineering and Industrial Technology. Also, it was deposited with the Fermentation Research Institute (IFO) under the accession number IFO 16335 on November 9, 1999.
Escherichia coli MM294 (DE3) / pLysS, pTB1516, which retains the plasmid pTB1516 used in Reference Example 1 described later, was established in 1992 by the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH). It has been deposited under the accession number FERM BP-3836 from May 21 and has been deposited under the accession number IFO15282 from April 16, 1992 (Heisei 4).
[0045]
【Example】
EXAMPLES The present invention will be described in more detail with reference to examples and reference examples below, but these do not limit the scope of the present invention.
[0046]
Example 1
Construction of 77-residue type (C-terminal 3 residue deletion type) betacellulin expression strain
A 77-residue (C-terminal 3-residue-deleted) betacellulin expression plasmid was constructed as follows [FIG. 1].
A 77-residue (C-terminal 3-residue-deleted) betacellulin structural gene is located adjacent to the upstream of the structural gene from the betacellulin expression plasmid pBO41 [Seno et al; Growth Factors, 13: 181 (1996)]. Amplified by PCR using Primer 1 (5′-CATATGATGGGGAATTCCACCAGAAGTCCTTG) with Nde I cleavage site and initiation codon, and Primer 2 (5′-GATCCCTAGTAGCAACTCTCTCACACTCTTGCTCC) with stop codon and Bam HI cleavage site after 77th aspartic acid did. The gene amplified by PCR was ligated to the pCR2.1 vector using TA original cloning kit (manufactured by Invitrogen) to prepare pCR2.1 / BTC77. This was introduced into Escherichia coli JM109, and a transformant was selected using ampicillin resistance and β-galactosidase activity as indicators. A transformant having pCR2.1 / BTC77 was cultured and pCR2.1 / BTC77 was prepared using QIAprep8 Miniprep kit (Qiagen).
pBRdesNde lacking the NdeI recognition site was prepared by cleaving pBR322 with NdeI, blunting the ends with T4 DNA polymerase (DNA Blunting Kit, manufactured by Takara Shuzo Co., Ltd.), and religating. pET3c was cleaved with Bgl II-Eco RV, and a fragment of about 0.26 kbp was recovered. The ends were blunted with T4 DNA polymerase, and ligated with the Sca I fragment of pBRdesNde to prepare pBR / T7 desNde. Moreover, pBR322desBam in which the BamHI recognition site of pBR322 was deleted was prepared by site-directed mutagenesis (Quick Change, manufactured by STRATAGENE). The Sph I-Eco RV fragment of pBR322desBam was ligated with the Sph I-Eco RV fragment of pBR / T7 desNde to prepare a tetracycline resistant expression vector pTCII. pCR2.1 / BTC77 was cleaved with NdeI and BamHI and subjected to agarose electrophoresis, and an approximately 240 bp 77-residue betacellulin structural gene was recovered using QIAquick Spin Purification Kit (Qiagen). The expression vector pTCII was cleaved with Nde I and Bam HI and subjected to agarose electrophoresis. Similarly, a band of about 4.6 kbp was recovered. After ligating the 77-residue betacellulin structural gene with the Nde I-Bam HI fragment of the expression vector pTCII, it was introduced into Escherichia coli JM109, a transformant was selected with tetracycline resistance, and the plasmid was recovered from the strain again. The expression plasmid was pTCIIBTC77.
This pTCIIBTC77 was introduced into Escherichia coli MM294 (DE3), a transformant was selected with resistance to tetracycline, and a 77-residue betacellulin-expressing strain MM294 (DE3) / pTCIIBTC77 was obtained.
[0047]
Example 2
Culture of 77-residue betacellulin-expressing strain
77-residue betacellulin-expressing strain MM294 (DE3) / pTCIIBTC77 in LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 10 mg / L tetracycline for 16 hours at 30 ° C. Cultured. 150 mL of the obtained culture broth was used as a main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.024% Magnesium sulfate, 0.02% Newpol LB-625, 0.0005% Thiamine hydrochloride, 1.5% glucose, 1.0% casamino acid, 1.0% yeast extract) 2L jar charged with 1.5 liters After transplantation to a fermenter, aeration and agitation culture was started at 37 ° C., an aeration rate of 2 L / min, and an agitation speed of 500 rpm. When the turbidity of the culture reached about 1200 kret units, 5.95 mg / L isopropyl-β-D-thiogalactopyranoside (IPTG) was added. When IPTG was added, 0.75% glucose was added 1,2.5 hours after the addition, and the culture was continued until 9 hours after the start of the culture. The culture solution was centrifuged at 10,000 rpm for 30 minutes to collect bacterial cells.
[0048]
Example 3
Purification of Met-77 residue betacellulin
After adding 10 mL of 0.1 M Tris-HCl (pH 8.0) containing 1 mM EDTA, 1 mM APMSF and 7 M guanidine hydrochloride to 5 g of the microbial cells obtained in Example 2, the mixture was stirred overnight at 4 ° C. and extracted. And centrifugation (10000 rpm, 20 minutes). To 10 mL of the obtained supernatant, 250 mL of 50 mM Tris-HCl (pH 8.0) containing 0.5 mM oxidized glutathione, 1 mM reduced glutathione, 1 mM EDTA, 0.1 M arginine hydrochloride and 2 M urea was added at 4 ° C. After overnight refolding. Centrifugation (10000 rpm, 20 minutes) was performed to obtain 260 mL of a centrifugal supernatant. The centrifugal supernatant was concentrated using a YM3 membrane (fractionated molecular weight: 3000, Millipore). 120 mL of 2M urea was added to 80 mL of concentrated desalted solution, and then adjusted to pH 5.0 with hydrochloric acid. Centrifugation (10000 rpm, 20 minutes) was performed, and the obtained supernatant was equilibrated with an SP-Toyopearl 650M column (2.2 cm × 12 cm, Tosoh Corporation) equilibrated with 50 mM sodium acetate buffer (pH 5.0). After adsorbing at a flow rate of 10 mL per minute and thoroughly washing with the buffer used for equilibration, elution was carried out with a sodium chloride linear concentration gradient from 0 M to 1.0 M. Fractions containing Met-77 residue type betacellulin were collected, and 1/3 of the fraction was adsorbed onto a C4P-50 column (1.0 cm × 25 cm, Showa Denko) equilibrated with 0.1% trifluoroacetic acid. Met-77 residue betacellulin was eluted with a linear gradient of acetonitrile from 13.5% to 21.2%. The eluate obtained by carrying out the same operation twice more was dialyzed against 0.02% trifluoroacetic acid and freeze-dried. The freeze-dried powder was dissolved in 10 mL of distilled water, passed through an acetic acid-type AG1-X8 column (1.0 cm × 10 cm, Nippon Bio-Rad), and then freeze-dried again to form Met-77 residue betacellulin. 8 mg was obtained.
[0049]
Example 4
Construction of 76-residue (C-terminal 4-residue-deleted) betacellulin expression plasmid
A 76-residue (C-terminal 4-residue-deleted) betacellulin expression plasmid was constructed as follows [FIG. 2].
Primer having a 76-residue (C-terminal 4-residue-deleted) betacellulin structural gene adjacent to the upstream of the structural gene from pTCII / BTC77 constructed in Example 1 and having an initiation codon PCR was performed using 1 (5′-CATATGATGGGGAATTCCACCCAGAAGTCTG) and primer 2 (5′-GGATCCCTAAACTCTCTCACACTTGTCCCAATG) having a stop codon and a Bam HI cleavage site after the 76th valine. The gene amplified by PCR was ligated to the pCR2.1 vector using TA original cloning kit (manufactured by Invitrogen) to prepare pCR2.1 / BTC76. This was introduced into Escherichia coli JM109, and a transformant was selected using ampicillin resistance and β-galactosidase activity as indicators. A transformant having pCR2.1 / BTC76 was cultured, and pCR2.1 / BTC76 was prepared using QIAprep8Miniprep kit (manufactured by Qiagen).
pCR2.1 / BTC76 was cleaved with Nde I and Bam HI and subjected to agarose electrophoresis, and about 240 bp of 76-residue betacellulin structural gene was recovered using QIAquick Spin Purification Kit (Qiagen). The expression vector pTCII prepared in Example 1 was cleaved with Nde I and Bam HI and subjected to agarose electrophoresis. Similarly, a band of about 4.6 kbp was recovered. After ligating the 76-residue betacellulin structural gene with the Nde I-Bam HI fragment of the expression vector pTCII, it was introduced into Escherichia coli JM109, a transformant was selected for tetracycline resistance, and the plasmid was recovered from the strain again. The expression plasmid was pTCIIBTC76.
This pTCIIBTC76 was introduced into Escherichia coli MM294 (DE3), a transformant was selected with resistance to tetracycline, and a 76-residue betacellulin-expressing strain MM294 (DE3) / pTCIIBTC76 was obtained.
[0050]
Example 5
Culture of 76-residue betacellulin-expressing strain
76-residue betacellulin-expressing strain MM294 (DE3) / pTCIIBTC76 in LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 10 mg / L tetracycline for 16 hours at 30 ° C. Cultured. The obtained culture broth was used as the main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.024% sulfuric acid. Magnesium, 0.02% Newpol LB-625, 0.0005% Thiamine hydrochloride, 1.5% glucose, 1.0% casamino acid, 1.0% yeast extract) transplanted to a 50L fermentor containing 20 liters Then, aeration and agitation culture was started at 37 ° C., an aeration rate of 20 L / min, and an agitation speed of 210 rpm. When the turbidity of the culture reached about 1200 kret units, 5.95 mg / L isopropyl-β-D-thiogalactopyranoside (IPTG) was added. When IPTG was added, 0.75% glucose was added 2 and 3.5 hours after the addition, and the culture was continued until 11 hours after the start of the culture. The culture solution was centrifuged at 10,000 rpm for 30 minutes, and 540 g of bacterial cells were collected.
[0051]
Example 6
Purification of Met-76 residue betacellulin
To 375 g of the bacterial cells obtained in Example 5, 1.0 L of 0.1 M Tris-HCl (pH 8.0) containing 1 mM EDTA, 1 mM APMSF and 7 M guanidine hydrochloride was added, followed by extraction at 4 ° C. overnight. Then, centrifugation (10000 rpm, 20 minutes) was performed. To 1.0 L of the obtained supernatant, 19 L of 50 mM Tris-HCl (pH 8.0) containing 0.5 mM oxidized glutathione, 1 mM reduced glutathione, 1 mM EDTA, 0.1 M arginine hydrochloride and 2 M urea was added. After refolding overnight at 0 ° C., centrifugation (10000 rpm, 20 minutes) was performed to obtain 20 L of a centrifugal supernatant. The centrifugal supernatant was concentrated using a Pellicon cassette system (fractionated molecular weight: 5000, Millipore). After adding 13.2 L of 2M urea to 3.3 L of concentrated desalted solution, the pH was adjusted to 5.0 with hydrochloric acid. After adsorbing to a POROS 50HS column (2.2 cm × 12 cm, Nippon Perceptive) equilibrated with 50 mM sodium acetate buffer (pH 5.0) at a flow rate of 30 mL / min and thoroughly washing with the buffer used for equilibration The elution was performed with a linear sodium chloride concentration gradient from 0.3 M to 1.3 M. Fractions containing 76-residue betacellulin were collected, diluted 3-fold with distilled water, and then equilibrated with 50 mM sodium acetate buffer (pH 4.5) (TSKgel CM-5PW column (2.15 cm × 15 cm, Added to Tosoh Corporation). The TSKgel CM-5PW column adsorbed with Met-76 residue betacellulin was eluted with a sodium chloride linear gradient from 0.24M to 0.44M. Fractions containing Met-76 residue betacellulin were collected and adsorbed onto a TSKgel ODS-120T column (2.15 cm × 30 cm, Tosoh Corp.) equilibrated with 0.1% trifluoroacetic acid, 17% to 24% Met-76 residue betacellulin was eluted with a linear gradient of acetonitrile. The eluate was dialyzed against 0.02% trifluoroacetic acid and lyophilized. The freeze-dried powder was dissolved in 10 mL of distilled water, passed through an acetic acid-type AG1-X8 column (1.0 cm × 10 cm, Nippon Bio-Rad), and then freeze-dried again to form Met-76 residue betacellulin. 93 mg was obtained.
[0052]
Example 7
Determination of the characteristics of betacellulin variants
The characteristics of Met-77 residue type betacellulin obtained in Example 3 and Met-76 residue type betacellulin obtained in Example 6 were determined as follows.
a) Analysis using SDS-polyacrylamide electrophoresis
The betacellulin variant and Met-80 residue betacellulin prepared by the method described in JP-A-10-191989 were prepared using a sample buffer (125 mM Tris-HCl, 1% sodium dodecyl sulfate, 15% glycerol, 5% 2-mercapto). (Ethanol, 0.005% bromophenol blue) and electrophoresed with Multigel 15/25 (Daiichi Kagaku). When the gel after electrophoresis was stained with Rapid CBB KANTO (Kanto Chemical Co., Inc.), all were almost single bands [FIG. 3].
b) Amino acid composition analysis
The betacellulin variant was subjected to gas phase hydrolysis with 6N hydrochloric acid containing 4% thioglycolic acid at 110 ° C. for 24 and 48 hours, and the amino acid composition was determined using an amino acid analyzer (Hitachi L-8500Amino Acid Analyzer). As a result, all the variants contained methionine derived from the start codon ATG, which was consistent with the amino acid composition predicted from the cDNA base sequence [Tables 1 and 2].
[0053]
[Table 1]

[0054]
[Table 2]

[0055]
c) N-terminal amino acid sequence analysis
N-terminal amino acid sequence analysis was determined using a gas phase protein sequencer (Applied Biosystems model 477A). As a result, all the variants were in agreement with the amino acid composition predicted from the cDNA base sequence. All of the variants had methionine derived from the start codon ATG at the N-terminus as in the 80-residue type (Tables 3 and 4).
[0056]
[Table 3]

[0057]
[Table 4]

[0058]
d) C-terminal amino acid analysis
The C-terminal amino acid was determined by the gas phase hydrazine decomposition method (100 ° C., 6 hours) using an amino acid analyzer (Hitachi L-8500 AAmino Acid Analyzer). Aspartic acid was detected in Met-77 residue type betacellulin, and valine was detected in Met-76 residue type betacellulin [Table 5, Table 6].
[Table 5]

[Table 6]

[0059]
Example 8
Measurement of proliferation promoting activity using 3T3 cells
Into the stationary 3T3 A31-714 clone 4 (International Journal of Cancer, Vol. 12, 463 (1973)) as described in Molecular Cell Biology, Vol. 8, 588 (1988). ^Three Growth-promoting activity was measured by H-thymidine incorporation.
That is, using Dulbecco's modified Eagle's MEM medium containing 5% bovine serum, 3T3 A31-714 clone 4 suspended at 1000 cells / mL was seeded in 100 μL each in a 96-well plate and placed in a carbon dioxide incubator (5% The cells were cultured at 37 ° C. for 1 day in carbon dioxide (95% air). 75 μL of the supernatant was extracted, and the serum concentration was adjusted to 1% by adding 100 μL of Dulbecco's modified Eagle MEM medium without serum. After further culturing for 2 days, Met-77 residue type betacellulin prepared in Example 3 at various concentrations, Met-76 residue type betacellulin prepared in Example 6, and JP-A-10-191989 Met-80 residue type betacellulin prepared by the method was added. 16 hours after addition ^Three 0.25 μCi / well of H-thymidine [Amersham Pharmacia Biotech] was added, and after 4 hours, the cells were washed 3 times with PBS, and then 100 μL of 5% SDS was added to lyse the cells. Transfer the cell lysate to a scintillation vial, add 1 mL of scintillator A (Wako Pure Chemical Industries, Ltd.), and use a scintillation counter. ^Three The uptake of H-thymidine into cells was measured [FIG. 4].
[0060]
Example 9
Measurement of β cell differentiation promoting activity using AR42J cells
Met- prepared in Example 3 was a pancreatic cancer-derived cell line AR42J [Chiristophe; American Journal of Physiology, 266: G963 (1994)] induced by a chemical carcinogen. 77-residue betacellulin, Met-76-residue betacellulin prepared in Example 6 or Met-80-residue betacellulin and 10% fetal bovine serum prepared by the method described in JP-A-10-191989 10 containing Dulbecco's modified Eagle MEM medium ^Five The cells were suspended to a cell / mL, 500 μL was seeded on a chamber slide, and cultured at 37 ° C. for 5 days in a carbon dioxide incubator (5% carbon dioxide, 95% air). After 5 days, the cells were washed once with PBS, fixed with 10% formaldehyde, treated with 0.1% Triton X-100 for 5 minutes, then added with Block Ace (Snow Brand, Japan) and blocked at room temperature for 40 minutes. Went. Anti-insulin antibody (advanced immunochemical) diluted with 10% Block Ace was added and reacted at room temperature for 40 minutes. 0.1% Triton X-100 was added and allowed to stand at room temperature for 5 minutes, and then washed 3 times with PBS. FITC (Fluorescein Isothiocyanate) -labeled anti-mouse IgG antibody (Cappel) diluted with 10% Block Ace was added and reacted at room temperature for 40 minutes. 0.1% Triton X-100 was added and allowed to stand at room temperature for 5 minutes, then washed 3 times with PBS, and observed with a fluorescence microscope. When any betacellulin was added, cells that were fluorescently stained, that is, cells differentiated into β-cells producing insulin were observed [FIG. 5].
[0061]
Example 10
Construction of human placental alkaline phosphatase gene expression vector
Differentiation-promoting activity into β cells was also performed using AR42J cells transformed with alkaline phosphatase linked as a reporter downstream of the insulin promoter. That is, cells differentiated into β-cells by betacellulin produce alkaline phosphatase, and the activity of promoting differentiation into β-cells can be quantitatively measured by measuring the activity of this alkaline phosphatase.
Genomic DNA was prepared from the rat tail according to a conventional method. Using this DNA as a template, primers RI-1 [5′-AGAGTCCAAGATCCCCCAACCACT-3 ′] and RI-3 [5 ′ synthesized based on the previously reported nucleotide sequence of the rat insulin II gene promoter (GenBank: Accession No. J00748) -AGCTGGTCACTTAGGGCTGGGG-3 ′] was used to amplify the insulin promoter region 0.75 Kb by the PCR method. Furthermore, PCR was performed using the PCR product as a template and using primers RI-1Cla [5′-GAATCGATATAGTCAAGGATCCCCA-3 ′] and RI-3Xho [5′-GACTCGAGCTGGTCACTTAGGG-3 ′]. The amplified 0.75 Kb DNA fragment was isolated and the base sequence of the cloned fragment was determined using the plasmid pTB1881 obtained by integration into the pT7Blue vector (Novagen 69820-1) and confirmed to be the rat insulin promoter. did. Plasmid pTB1881 was digested with Xho I-Cla I to obtain a 0.73 kb DNA fragment which is a rat insulin promoter. Next, an expression plasmid pTB1330 of a 2.0 kb cDNA (J. Berger et al., Gene 66, 1 (1988)) encoding human placental alkaline phosphatase (PLAP) was digested with Xho I-Hind III. A 7 kb DNA fragment (including PLAP cDNA, SV40-derived splicing site and poly A addition site, pBR322-derived ori and ampicillin resistance gene) was isolated, and the aforementioned rat insulin promoter region 0.73 kb Xho I-Cla I fragment was subjected to T4 DNA ligase reaction. To obtain plasmid pTB1898.
[0062]
Example 11
Construction of PLAP-expressing AR42J cells
Placing PLAP expression plasmids pTB1898 and Tn5 neo in AR42J cells ^r Plasmid pMCneopoly A (STRATAGENE) containing the gene is transfected with TransIT ^TM -Simultaneous introduction using LT1 (Mirus, PenVera Corporation). After the introduction, the cells were cultured in DMEM supplemented with 10% fetal bovine serum for 2 days, and then added to a selective medium supplemented with 800 μg / mL G418 (Genethecin, GIBCO BRL), and the culture was continued. Clones were isolated from cells that had grown to be G418 resistant by the limiting dilution method.
Cells of each clone were seeded in a 24-well plate, cultured for 4 days with and without addition of 20 ng / mL of Met-80 residue betacellulin, and the culture supernatant was collected and heat-treated at 65 ° C. for 30 minutes, Alkaline phosphatase activity in the medium was measured. A clone was selected that showed an increase in alkaline phosphatase activity upon addition of 80-residue betacellulin. The results of several clones are shown in [Table 7] below.
[Table 7]

[0063]
Example 12
Measurement of β-cell differentiation promoting activity using PLAP-expressing AR42J cells
PLAP-expressing AR42J cells constructed in Example 11 were prepared from various concentrations of Met-77 residue-type betacellulin prepared in Example 3, Met-76 residue-type betacellulin prepared in Example 6, or JP-A-10-191989. 10 using Dulbecco's modified Eagle MEM medium containing Met-80 residue betacellulin and 10% fetal bovine serum prepared by the method described in No. ^Five The cells were suspended to a cell / mL, 100 μL was seeded in a 96-well plate, and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% air) at 37 ° C. for 5 days. After 5 days, the culture supernatant was sampled and treated at 65 ° C. for 30 minutes, and then 50 μL of 50 μL of 2 × SEAP (2M diethanolamine, 1 mM MgCl ₂ , 20 mM homoarginine) was added to a 96-well microplate. After maintaining at 37 ° C. for 10 minutes, 10 μL of 20 mg / mL p-nitrophenyl phosphoric acid (Sigma) was added and reacted at 37 ° C. for 16 hours [FIG. 6]. Met-77 residue type betacellulin and Met-76 residue type betacellulin exhibited almost the same differentiation-inducing promoting activity as Met-80 residue type betacellulin.
[0064]
Example 13
Construction of 2-cell residue type (N-terminal 1 residue, C-terminal 3 residue deletion type) betacellulin expression strain
A 2-76 residue type (N-terminal 1 residue, C-terminal 3 residue deletion type) betacellulin expression plasmid was constructed as follows [FIG. 7].
2-cell residue type (N-terminal 1 residue, C-terminal 3 residue deletion type) betacellulin structural gene was converted into a 76-residue type (C-terminal 3 residue deletion type) betacellulin expression plasmid pTCIIBTC76 From Example 4], primer 3 (5′-CAGCATATGGGGATTCCACCAGAAGTCCT) having an Nde I cleavage site and a start codon adjacent to the upstream of the second glycine, and a primer having a stop codon and a Bam HI cleavage site after the C-terminal valine 4 (5′-GGATCCCTAAACTCTCTCACACCTTGCTCCAATG) was used to amplify by PCR. The gene amplified by PCR was ligated to the pCR2.1 vector using TA original cloning kit (manufactured by Invitrogen) to prepare pCR2.1BTC2-76. This was introduced into Escherichia coli JM109, and transformants were selected using ampicillin resistance as an index. A transformant having pCR2.1BTC2-76 was cultured, and pCR2.1BTC2-76 was prepared using QIAprep8 Miniprep kit (Qiagen).
pCR2.1BTC2-76 was cleaved with Nde I and Bam HI and subjected to agarose electrophoresis, and an approximately 230 bp 2-76 residue betacellulin structural gene was recovered using QIAGEN gel extraction kit (Qiagen). . The expression vector pTCII [Example 1] was cleaved with Nde I and Bam HI and subjected to agarose electrophoresis, and a band of about 4.6 kbp was similarly collected. After ligating the 2-76-residue betacellulin structural gene with the Nde I-Bam HI fragment of the expression vector pTCII, it was introduced into Escherichia coli JM109, a transformant was selected for resistance to tetracycline, and the plasmid was recovered from that strain again. The expression plasmid was pTCIIBTC2-76.
This pTCIIBTC2-76 was introduced into Escherichia coli MM294 (DE3), and a transformant was selected with resistance to tetracycline to obtain a 2-76 residue betacellulin expression strain MM294 (DE3) / pTCIIBTC2-76.
[0065]
Example 14
Culture of 2-76-residue betacellulin-expressing strain
30-mL of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 10 mg / L tetracycline of 2-76-residue betacellulin-expressing strain MM294 (DE3) / pTCIIBTC2-76 The culture was performed at 0 ° C. for 16 hours. The obtained culture broth was used as the main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.012% sulfuric acid. Magnesium, 0.0007% thiamine hydrochloride, 1.5% dextrose, 1.5% high case) Grafted into 6 ml of 1L Meyer containing 200mL and started agitation culture at 37 ° C and agitation speed of 200rpm did. When the turbidity of the culture reached about 160 kret units, 5.95 mg / L isopropyl-β-D-thiogalactopyranoside (IPTG) was added. Culturing was performed until 4 hours after the addition of IPTG. The culture solution was centrifuged at 9000 rpm for 30 minutes to collect 5.1 g of bacterial cells.
[0066]
Example 15
Purification of 2-76 residue betacellulin
After adding 15 mL of 0.1 M Tris-HCl (pH 8.0) containing 1 mM EDTA, 1 mM APMSF and 7 M guanidine hydrochloride to 5 g of the microbial cell obtained in Example 14, the mixture was stirred overnight at 4 ° C. and extracted. And centrifugation (9000 rpm, 10 minutes). To 15 mL of the obtained supernatant, 250 mL of 50 mM Tris-HCl (pH 8.0) containing 0.5 mM oxidized glutathione, 1 mM reduced glutathione, 1 mM EDTA, 0.1 M arginine hydrochloride and 2 M urea was added at 4 ° C. After refolding overnight, centrifugation (10000 rpm, 10 minutes) was performed to obtain 265 mL of a centrifugal supernatant. 1 L of 2M urea was added to the centrifugal supernatant, adjusted to pH 5.0 with acetic acid, and allowed to stand at 4 ° C. overnight. Centrifugation (10000 rpm, 10 minutes) was performed, and the obtained supernatant was equilibrated on a POROS 50HS column (2.2 cm × 12 cm, Nippon Perceptive) equilibrated with 50 mM sodium acetate buffer (pH 5.0) every minute. After adsorbing at a flow rate of 30 mL and thoroughly washing with the buffer used for equilibration, elution was performed with a 0.3 M to 1.3 M sodium chloride linear concentration gradient. Fractions containing 2-76-residue betacellulin were collected, diluted 3-fold with 50 mM sodium acetate buffer (pH 4.5), and then equilibrated with this buffer TSKgel CM-5PW column (0.75 cm X 7.5 cm, Tosoh Corporation). The TSKgel CM-5PW column adsorbed with 2-76 residue betacellulin was eluted with a 0.3 M to 0.5 M sodium chloride linear gradient. Fractions containing 2-76 residue betacellulin were collected and adsorbed onto a TSKgel ODS-120T column (2.15 cm × 30 cm, Tosoh Corp.) equilibrated with 0.1% trifluoroacetic acid, 20% to 44% The 2-76 residue betacellulin was eluted with a linear gradient of acetonitrile. The eluate was lyophilized, dissolved in 5 mL of distilled water, passed through an acetic acid-type AG1-X8 column (1.0 cm × 10 cm, Nippon Bio-Rad), and then lyophilized again to leave 2-76 residue. 0.7 mg of basic betacellulin was obtained.
[0067]
Example 16
Construction of 24-cell residue type (N-terminal 23 residues, C-terminal 3 residue deletion type) betacellulin expression strain
A 24-cell residue type (N-terminal 23 residues, C-terminal 3 residue deletion type) betacellulin expression plasmid was constructed as follows [FIG. 8].
24-76 residue type (N-terminal 23 residues, C-terminal 3 residue deletion type) beta-cellulin structural gene prepared in Example 4 76 residue type (C-terminal 3 residue deletion type) beta From the cellulin expression plasmid pTCIIBTC76, primer 5 (5′-CAGCATATGCTCACCACCACACAATCAAAG) having an Nde I cleavage site and a start codon adjacent to the upstream of the 24th alanine, and a stop codon and a Bam HI cleavage site after the C-terminal valine Amplification was performed by PCR using primer 4 (5′-GGATCCCTAAACTCTCTCACACTTGTCCCAATG). The gene amplified by PCR was ligated to the pCR2.1 vector using TA original cloning kit (manufactured by Invitrogen) to prepare pCR2.1BTC24-76. This was introduced into Escherichia coli JM109, and transformants were selected using ampicillin resistance as an index. A transformant having pCR2.1BTC24-76 was cultured, and pCR2.1BTC24-76 was prepared using QIAprep8 Miniprep kit (manufactured by Qiagen).
pCR2.1BTC24-76 was cleaved with Nde I and Bam HI and subjected to agarose electrophoresis, and an approximately 160 bp 24-76 residue type betacellulin structural gene was recovered using QIAGEN gel extraction kit (Qiagen). The expression vector pTCII prepared in Example 1 was cleaved with Nde I and Bam HI and subjected to agarose electrophoresis. Similarly, a band of about 4.6 kbp was recovered. After ligating the 24-76-residue betacellulin structural gene with the Nde I-Bam HI fragment of the expression vector pTCII, it was introduced into Escherichia coli JM109, a transformant was selected for resistance to tetracycline, and the plasmid was recovered again from that strain. The expression plasmid was pTCIIBTC24-76.
This pTCIIBTC24-76 was introduced into Escherichia coli MM294 (DE3), a transformant was selected with resistance to tetracycline, and a 24-76 residue betacellulin expression strain MM294 (DE3) pTCIIBTC24-76 was obtained.
[0068]
Example 17
Culturing of 24-cell residue type betacellulin expression strain
30 to 30 mL of LB medium (1% peptone, 0.5% yeast extract, 0.5% sodium chloride) containing 10 mg / L tetracycline of the 24-76 residue betacellulin expression strain MM294 (DE3) / pTCIIBTC24-76 The culture was performed at 0 ° C. for 16 hours. The obtained culture broth was used as the main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.1% ammonium chloride, 0.05% sodium chloride, 0.012% sulfuric acid. (Magnesium, 0.0007% thiamine hydrochloride, 1.5% glucose, 1.5% high case) transplanted to five 1 L Mayer containing 150 mL, and started stirring culture at 37 ° C. and stirring speed of 200 rpm. When the turbidity of the culture reached about 160 kret units, 5.95 mg / L isopropyl-β-D-thiogalactopyranoside (IPTG) was added. Culturing was performed until 4 hours after the addition of IPTG. The culture solution was centrifuged at 10,000 rpm for 30 minutes to collect 3.9 g of bacterial cells.
[0069]
Example 18
Purification of 24-cell residue betacellulin
To 3.9 g of the microbial cells obtained in Example 5, 12 mL of 0.1 M Tris-HCl (pH 8.0) containing 1 mM EDTA, 1 mM APMSF and 7 M guanidine hydrochloride was added, followed by extraction at 4 ° C. overnight. Thereafter, centrifugation (9000 rpm, 10 minutes) was performed. To 12 mL of the obtained supernatant, 200 mL of 50 mM Tris-HCl (pH 8.0) containing 0.5 mM oxidized glutathione, 1 mM reduced glutathione, 1 mM EDTA, 0.1 M arginine hydrochloride and 2 M urea was added at 4 ° C. After refolding overnight, centrifugation (10000 rpm, 10 minutes) was performed to obtain 212 mL of a centrifugal supernatant. After adding 0.8 L of 2M urea to this centrifugal supernatant, it was adjusted to pH 5.0 with acetic acid and allowed to stand at 4 ° C. overnight. Centrifugation (10000 rpm, 10 minutes) was performed, and the obtained supernatant was equilibrated on a POROS 50HS column (2.2 cm × 12 cm, Nippon Perceptive) equilibrated with 50 mM sodium acetate buffer (pH 5.0) every minute. After adsorbing at a flow rate of 30 mL and thoroughly washing with the buffer used for equilibration, elution was performed with a 0.3 M to 1.3 M sodium chloride linear concentration gradient. Fractions containing 24-76-residue betacellulin were collected, diluted 3-fold with 50 mM sodium acetate buffer (pH 4.5), and then equilibrated with this buffer, a TSKgel CM-5PW column (0.75 cm × 7.5 cm, Tosoh Corporation). The TSKgel CM-5PW column adsorbed with 24-76 residue betacellulin was eluted with a 0.3 M to 0.5 M sodium chloride linear gradient. Fractions containing 24-76 residue betacellulin were collected and adsorbed onto a TSKgel ODS-120T column (2.15 cm × 30 cm, Tosoh) equilibrated with 0.1% trifluoroacetic acid, 20% to 44% The 24-cell residue type betacellulin was eluted with an acetonitrile linear concentration gradient. The eluate was freeze-dried, dissolved in 5 mL of distilled water, passed through an acetic acid-type AG1-X8 column (1.0 cm × 10 cm, Nippon Bio-Rad), and then freeze-dried again to leave 24-76 residue. 0.4 mg of basic betacellulin was obtained.
[0070]
Example 19
Determination of the characteristics of betacellulin variants
The characteristics of 2-76 residue type betacellulin obtained in Example 3 and 24-76 residue type betacellulin obtained in Example 6 were determined as follows.
a) Analysis using SDS-polyacrylamide electrophoresis
The betacellulin variant and the 76-residue betacellulin prepared in Example 6 were mixed with a sample buffer (125 mM Tris-HCl, 1% sodium dodecyl sulfate, 15% glycerol, 5% 2-mercaptoethanol, 0.005% bromophenol blue. ), And electrophoresis was performed with Multigel 15/25 (first chemical). When the gel after electrophoresis was stained with Rapid CBB KANTO (Kanto Chemical Co., Inc.), all were almost single bands [FIG. 9].
b) Amino acid composition analysis
The betacellulin variant was subjected to gas phase hydrolysis with 6N hydrochloric acid containing 4% thioglycolic acid at 110 ° C. for 24 and 48 hours, and amino acid composition was determined using an amino acid analyzer (Hitachi L-8500A Amino Acid Analyzer). did. As a result, all the variants were in agreement with the amino acid composition predicted from the cDNA base sequence [Tables 8 and 9].
[0071]
[Table 8]

[0072]
[Table 9]

[0073]
c) N-terminal amino acid sequence analysis
N-terminal amino acid sequence analysis was determined using a gas phase protein sequencer (Applied Biosystems model 477A). As a result, all the variants were in agreement with the amino acid sequence predicted from the cDNA base sequence [Tables 10 and 11].
[0074]
[Table 10]

[0075]
[Table 11]

[0076]
d) C-terminal amino acid analysis
The C-terminal amino acid was determined by the gas phase hydrazine decomposition method (100 ° C., 6 hours) using an amino acid analyzer (Hitachi L-8500A Amino Acid Analyzer). In all the variants, valine was detected [Table 12, Table 13].
[Table 12]

[Table 13]

[0077]
Example 20
1) EGF binding assay using A431 cells
Human squamous cell carcinoma cell A431 cells, which are EGF receptor high expression strains, were used. ¹²⁵ Cell growth promoting activity was measured by binding inhibitory activity against I-EGF.
That is, using Dulbecco's Modified Eagle MEM (DMEM) medium containing 10% fetal bovine serum, ^Five A431 cells suspended to a cell / mL were seeded in 100 μL each in a 96-well plate and cultured at 37 ° C. for 2 days in a carbon dioxide incubator (5% carbon dioxide, 95% air). Two days later, after washing three times with 200 μL / well of a binding medium containing 20 mM HEPES and 0.1% bovine serum albumin mixed in equal amounts of DMEM and F-12, various concentrations of unlabeled EGF, Example 15 2-76 residue betacellulin prepared in Example 18, 24-76 residue betacellulin prepared in Example 18, Met-76 residue betacellulin prepared in Example 6, or JP-A-10-191989. Met-80 residue betacellulin and 0.25 nM prepared by the described usage ¹²⁵ 100 μL of binding medium containing I-EGF (Amersham Pharmacia Biotech) was added. After standing at 4 ° C. for 90 minutes, the plate was washed 3 times with 200 μL of binding medium, and the cells were lysed with 200 μL of 0.1N aqueous sodium hydroxide solution containing 1% SDS. Transfer the cell lysate to a scintillation vial, add 1 mL of scintillator A (Wako Pure Chemical Industries, Ltd.), and use a scintillation counter. ¹²⁵ The binding inhibitory activity against I-EGF was measured.
2) Measurement of β-cell differentiation promoting activity using human placental alkaline phosphatase-expressing AR42J cells
PLAP-expressing AR42J cells were prepared from various concentrations of 2-76 residue betacellulin prepared in Example 15, 24-76 residue betacellulin prepared in Example 18, and Met-76 residue prepared in Example 6. Type betacellulin or Dulbecco's modified Eagle's MEM medium containing Met-80 residue betacellulin and 10% fetal bovine serum prepared by the method described in JP-A-10-191989. ^Five The cells were suspended to a cell / mL, 100 μL was seeded in a 96-well plate, and cultured for 5 days at 37 ° C. in a carbon dioxide incubator (5% carbon dioxide, 95% air). After 5 days, the culture supernatant was sampled and treated at 65 ° C. for 30 minutes, and then 50 μL was added in advance to 50 μL of 2 × SEAP (2M diethanolamine, 1 mM MgCl 2 ₂ , 20 mM homoarginine) was added to a 96-well microplate. 10 μL of 20 mg / mL p-nitrophenyl phosphoric acid (Sigma) was added and reacted at 37 ° C. for 16 hours. In all the variants, the ratio of EGF activity to BTC activity decreased to about 5% of Met-80 residue type betacellulin, which was the same as that of Met-76 residue type betacellulin, and the effect of N-terminal deletion was observed. I couldn't.
3) The results of 1) and 2) are summarized in [Table 14].
[Table 14]

[0078]
Example 21
Construction of hBTC50 modified molecule A expression plasmid pTB1985 in which three residues derived from hereregulin (Her) are inserted between Cys3-Cys4
Using pTB1976 as a template, (1) 5′-side primer PET-1: 5′-GAAATAATTTTGTTTACTACTTAAGAAGGGAG-3 ′ and 3′-side primer BTC-1: 5′-AGGAGGGGCGTCGAGGGGGTCTGCTCGCGCCA-3 ′, (2) 5′-side primer BTC-2 : 5'-TGGCCGAGCAGAACCCCTCGACGCCCCTCCT-3 'and 3'-side primer BTC-3: 5'-TCTATGCGCACCCGTTCTCGAGCACTGTTC-3' was used for PCR.
Next, PCR was performed using the mixture of the PCR products of (1) and (2) as a template and using the 5 ′ primer BT-95h and the 3 ′ primer BT-94h, and the sequence of Her between Cys3-Cys4 of hBTC50 A DNA fragment encoding modified molecule A in which three amino acids (Asn, Pro, Ser) were inserted was obtained. This DNA fragment was digested with NdeI and BamHI and then incorporated into the NdeI-BamHI site of pET-3c to prepare pTB1985.
[0079]
Example 22
Construction of hBTC50 modified molecule B expression plasmid pTB1987 in which N-terminal 7 residues are replaced with 5 residues of epidermal growth factor (EGF) corresponding to the site
Using pTB1976 as a template, PCR was performed using 5 ′ primer BTC-7: 5′-TATACATAGAGAGCGCACTCTGAGTGCCCCAAGC-3 ′ and 3 ′ primer BT-94h, and 5 residues of EGF corresponding to the N-terminal 7 residues of hBTC50 Thus, a DNA fragment encoding the modified molecule B was substituted. This DNA fragment was digested with NdeI and BamHI and then incorporated into the NdeI-BamHI site of pET3c to prepare pTB1987.
[0080]
Example 23
Expression in E. coli
Under the control of the Φ10 promoter of T7 phage, plasmid pTB1976, plasmid pTB1985 and plasmid pTB1987 obtained in Reference Examples described later and in the above Examples were introduced into E. coli BL21 (DE3) / pLysS (Novagen) for expression. Each E. coli recombinant was cultured at 37 ° C. in LB medium containing 100 μg / ml ampicillin and 10 μg / ml chloramphenicol. At the time of the cret unit 160, isopropyl β-D-thiogalactopyranoside was added to a final concentration of 0.4 mM, and the culture was further continued at 37 ° C. for 4 hours. After incubation, the cells were collected by centrifugation and stored at −80 ° C. until extraction.
[0081]
Example 24
Purification of hBTC50, modified molecule A and modified molecule B
4-1) Preparation of E. coli inclusion bodies
The cells collected from 400 ml of the culture solution are suspended in 20 ml of 10 mM Tris-HCl (pH 8.0), 10% sucrose, 10 mM EDTA, frozen and thawed, and then placed in ice for 30 minutes to completely Cells were lysed. After sonication (10 sec, 3 times) under ice-cooling, the precipitate was collected again by centrifugation (9000 rpm, 15 min, 4 ° C .: Beckman). This washing operation was repeated three times to prepare inclusion bodies.
4-2) Protein extraction and refolding
The inclusion body obtained in 4-1) was added to 8 ml of 7M guanidine-HCl, 0.1M Tris-CH. _Three The recombinant protein was extracted by suspending in COOH (pH 8.0), 1 mM EDTA, and gently stirring with a stirrer at 4 ° C. for 1 hour. Next, reduced glutathione was added to 0.1 M and the pH was adjusted to 8.4 with a NaOH solution, and then nitrogen gas was blown into the extract to replace the air. After standing at room temperature for 2 hours, 20 volumes of 10 mM Tris-CH _Three Diluted with COOH (pH 8.0), 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine. Oxidized glutathione was added to 0.5 mM to the supernatant after centrifugation (9000 rpm, 15 min, 25 ° C.) to remove insoluble matters, and the mixture was allowed to stand at room temperature for 16 hours. Subsequently, the centrifugation (9000 rpm, 15 min, 25 ° C.) supernatant was lyophilized and concentrated, followed by 50 mM Tris-CH at 4 ° C. _Three Dialyzed against COOH (pH 5.5) and 1 mM EDTA (Spectra por: MWCO 1,000). After dialysis, the supernatant was centrifuged (9000 rpm, 15 min, 4 ° C.) through a cellulose acetate membrane (Millex GV, 0.22 um: Millipore) to remove insoluble matters.
4-3) Protein purification
The solution containing the refolded protein was applied to a cation exchange HPLC column (250 × 4.1 mm Id, Synchrom CM300, Synchropak) equilibrated with 50 mM sodium acetate (pH 5.5), and a NaCl concentration of 0 to 1M was applied. Protein was eluted and fractionated with a gradient. OD ₂₈₀ Elution fractions monitored with 0.1% HCl, 1% CH _Three C equilibrated with CN _Four Reversed phase HPLC column (150 × 4.6 mm Id, Ultron 300 C _Four : Chromatopacking Center), 1-40% CH _Three Elute with a CN concentration gradient. Next, the peak fraction obtained by column chromatography was freeze-dried. The yields of modified molecules A and B were 78.7 μg and 103.1 μg, respectively.
4-4) Protein purity test
For the purpose of the purity test of the purified standard, C ₁₈ 15-30% CH over a reverse phase HPLC column (150 x 4.6 mm, ODS120T, Tosoh) _Three Elution was performed with a CN concentration gradient, and the singleness of the peak patterns of the modified molecules A and B was confirmed. It was found that the purity of both proteins was 94% or more.
[0082]
Example 25
Measurement of cell growth promoting activity using 3T3 cells
According to the method described in Molecular and Cellular Biology, 8, 588, 1988, 3T3 A31-714 clone 4 in a stationary state (International Journal of Cancer, 12, 463, 1973) ) Into ^Three Cell growth promoting activity was measured by uptake of H-thymidine.
Using a Dulbecco's modified Eagle MEM medium containing 5% bovine serum, 100 μL of 3T3 A31-714 clone 4 suspended at 1000 cells / mL is seeded in a 96-well plate in a carbon dioxide incubator (5% carbon dioxide). , 95% air) at 37 ° C. for 1 day. 75 μL of the supernatant was extracted, and the serum concentration was adjusted to 1% by adding 100 μL of Dulbecco's modified Eagle MEM medium without serum. After further culturing for 2 days, various concentrations of hBTC50, modified molecule A or modified molecule B prepared in the above examples were added. 16 hours after addition ^Three 0.25 μCi / well of H-thymidine [Amersham Pharmacia Biotech] was added, and after 4 hours, the cells were washed 3 times with PBS, and then 100 μL of 5% SDS was added to lyse the cells. Transfer the cell lysate to a scintillation vial, add 1 mL of scintillator A (Wako Pure Chemical Industries, Ltd.), and use a scintillation counter. ^Three The uptake of H-thymidine into cells was measured.
From this measurement result, the maximum uptake value of 80-residue betacellulin (standard product) is taken as 100%, and the concentration of hBTC50 and modified molecule (ED) when 50% activity is shown (ED ₅₀ ) Is shown in [Table 15].
[Table 15]

[0083]
Example 26
Measurement of β-cell differentiation inducing activity using AR42J cells
One type selected from hBTC50, modified molecule A, modified molecule B prepared above and 80-residue betacellulin (hBTC80) prepared by the method described in JP-A-10-191989, and 10% fetal bovine serum A pancreatic cancer-derived cell line AR42J induced by a chemical carcinogen [Chiristophe; American Journal of Physiol., 266, G963, 1994] in a Dulbecco's modified Eagle MEM medium containing 10 ^Five The cells were suspended to a cell / mL, 500 μL was seeded on a chamber slide, and cultured at 37 ° C. for 5 days in a carbon dioxide incubator (5% carbon dioxide, 95% air). After 5 days, the cells were washed once with PBS, fixed with 10% formaldehyde, treated with 0.1% Triton X-100 for 5 minutes, Block Ace (Snow Brand, Japan) was added, and room temperature was maintained for 40 minutes. Blocking was performed. Anti-insulin antibody (advanced immunochemical) diluted with 10% Block Ace was added and reacted at room temperature for 40 minutes. 0.1% Triton X-100 was added and allowed to stand at room temperature for 5 minutes, and then washed 3 times with PBS. FITC (Fluorescein Isothiocyanate) labeled anti-mouse IgG antibody (Cappel) diluted with 10% Block Ace was added and reacted at room temperature for 40 minutes. 0.1% Triton X-100 was added and allowed to stand at room temperature for 5 minutes, then washed 3 times with PBS, and observed with a fluorescence microscope.
As a result, in the same manner as when hBTC80 was added, when hBTC50, modified molecule A, and modified molecule B were added, cells that were fluorescently stained, that is, cells differentiated into β-cells producing insulin were observed.
In addition, there was no difference in the appearance frequency of insulin-producing cells among hBTC80, hBTC50, modified molecule A, and modified molecule B.
[0084]
Example 27
Measurement of β cell differentiation inducing activity using PLAP-expressing AR42J cells
3-1) Construction of human placental alkaline phosphatase gene expression vector
The differentiation promoting activity into β cells was also carried out using AR42J cells transformed with an alkaline phosphatase gene linked as a reporter downstream of the insulin promoter. That is, cells differentiated into β-cells by betacellulin produce alkaline phosphatase, and the activity of promoting differentiation into β-cells can be quantitatively measured by measuring the activity of this alkaline phosphatase.
Genomic DNA was prepared from the rat tail according to a conventional method. Primer RI-1: 5′-AGAGTCAAGGATCCCCCAACCACT-3 ′ and primer RI-3: 5 ′ synthesized based on the previously reported nucleotide sequence of the rat insulin II gene promoter (GenBank: Accession No. J00748) using this DNA as a template -Insulin promoter region 0.75 Kb was amplified by PCR using AGCTGGTCACTTAGGGCTGGGG-3 '. Furthermore, PCR was performed using this PCR product as a template and using a primer RI-1Cla: 5′-GAATCGATAGAGCCAAGGATCCCCA-3 ′ and a primer RI-3Xho: 5′-GACTCGAGCTGGTCACTTAGGG-3 ′. The amplified 0.75 Kb DNA fragment is isolated, the nucleotide sequence of the cloned fragment is determined using the plasmid pTB1881 obtained by integrating into the pT7Blue vector (Novagen 69820-1), and it is a rat insulin II promoter It was confirmed. Plasmid pTB1881 was digested with Xho I-Cla I to obtain a 0.73 kb DNA fragment which is a rat insulin promoter. Next, an expression plasmid pTB1330 of 2.0 kb cDNA (J. Berger et al., Gene 66, 1 (1988)) encoding human placental alkaline phosphatase (PLAP) was digested with Xho I-Hind III. A 7 kb DNA fragment (including PLAP cDNA, SV40-derived splicing site and poly A addition site, pBR322-derived ori and ampicillin resistance gene) was isolated, and the aforementioned rat insulin promoter region 0.73 kb Xho I-Cla I fragment was isolated from T4 DNA ligase. Ligation by reaction yielded plasmid pTB1898.
[0085]
3-2) Construction of PLAP-expressing AR42J cells
Placing PLAP expression plasmids pTB1898 and Tn5 neo in AR42J cells ^r Plasmid pMCneopoly A (STRATAGENE) containing the gene is transfected with TransIT ^TM -Simultaneous introduction using LT1 (Mirus, PenVera Corporation). The cells after introduction were cultured in DMEM supplemented with 10% fetal bovine serum for 2 days, and then the culture was continued in place of a selective medium supplemented with 800 μg / mL of G418 (Geneticin, GIBCO BRL). Clones were isolated from cells that had grown to be G418 resistant by the limiting dilution method.
Cells of each clone were seeded in a 24-well plate and cultured for 4 days with and without the addition of 20 ng / mL of 80-residue BTC. The culture supernatant was collected and heat-treated at 65 ° C. for 30 minutes, Alkaline phosphatase activity was measured. A clone in which an increase in alkaline phosphatase activity was observed when 80-residue betacellulin was added was selected, and one clone AR71-104 was used for the following examination.
3-3) Measurement of β-cell differentiation inducing activity using PLAP-expressing AR42J cells
PLAP expression constructed in 3-2) above in Dulbecco's modified Eagle MEM medium containing various concentrations of hBTC50 prepared above, modified molecule A and modified molecule B, and 10% fetal bovine serum 3x10 AR42J cells ^Four The cells were suspended to a cell / mL, 100 μL was seeded in a 96-well plate, and cultured in a carbon dioxide incubator (5% carbon dioxide, 95% air) at 37 ° C. for 5 days. After 5 days, the culture supernatant was sampled and treated at 65 ° C. for 30 minutes, and then 50 μL of 50 μL of 2 × SEAP (2M diethanolamine, 1 mM MgCl ₂ , 20 mM homoarginine) was added to a 96-well microplate. After maintaining at 37 ° C. for 10 minutes, 10 μL of 20 mg / mL p-nitrophenyl phosphoric acid (Sigma) was added and reacted at 37 ° C. for 16 hours. Absorbance A at 405 nm ₄₀₅ Was measured.
Protein concentration required to show 50% activity when the maximum absorbance at the time of hBTC50 addition is 100% (ED ₅₀ ) Is shown in [Table 16].
[Table 16]

[0086]
Reference example 1
Construction of 50-residue human betacellulin (hBTC50) expression plasmid pTB1976
Using plasmid pTB1516 (described in JP-A-6-87894; accession number FERM BP-3836, accession number IFO 15282) into which 80-residue hBTC cDNA has been incorporated as a template, primer BT-95h: 5'-AGCATATCGCGGAAAGCCCACTTCTCTAGGT-3 'and PCR was performed using BT-94h: 5′-CTGGATCCTAGTAAAAACAAGTCAACTCTCT-3 ′. A PCR product in which a translation initiation codon and an NdeI site were inserted at the 5 ′ end of the C-terminal 50 residue type of hBTC and a stop codon and a BamHI site were inserted at the 3 ′ end was digested with NdeI and BamHI, respectively, and the Φ10 promoter of T7 phage was The DNA Ligation Kit Ver. 2 (Takara) was used to construct the expression plasmid pTB1976 of hBTC50. The nucleotide sequence of the incorporated cDNA was confirmed by an ABI DNA sequencer (ABI377 DNA Sequencer).
An outline of the construction of plasmid pTB1976 is shown in FIG.
[0087]
【The invention's effect】
The betacellulin mutein of the present invention or a salt thereof is useful as an excellent therapeutic agent for diabetes because EGF activity is attenuated while maintaining BTC activity and there is no antigenic problem.
[0088]
[Sequence Listing]

[0089]
[Brief description of the drawings]
FIG. 1 shows the construction of a 77-residue type (C-terminal 3 residue deletion type) betacellulin expression plasmid.
FIG. 2 shows the construction of a 76-residue type (C-terminal 4-residue deletion type) betacellulin expression plasmid.
FIG. 3 shows a diagram showing the results of electrophoresis of betacellulin mutein performed in Example 7.
FIG. 4 was performed in Example 8 ^Three The figure which shows the uptake | capture result to the cell of H-thymidine is shown.
FIG. 5 shows a fluorescence micrograph of cells differentiated into β-cells described in Example 9.
6 shows the results of the β cell differentiation promoting activity performed in Example 12. FIG.
FIG. 7 shows the construction of a 2-76 residue type (N-terminal 1 residue, C-terminal 4 residue deletion type) betacellulin expression plasmid.
FIG. 8 shows the construction of an expression plasmid for 24-cell residue type (N-terminal 23 residues, C-terminal 4 residue deletion type) betacellulin.
FIG. 9 is a diagram showing the results of gel staining after electrophoresis performed in Example 19.
FIG. 10 shows an outline of the construction of plasmid pTB1976 described in Reference Example 1.

Claims (12)

In betacellulin comprising the amino acid sequence represented by SEQ ID NO: 35, 1 to 40 amino acid residues from the N terminus may be deleted, and the C terminus containing the third amino acid residue Leu from the C terminus to the amino acid sequence 3 or 4 amino acid residues are deleted from, held differentiation promoting activity of pancreatic β cells, epithelial cell growth promoting activity is reduced, betacellulin mutein or a salt thereof. The betacellulin mutein or a salt thereof according to claim 1, wherein the ratio of the differentiation promoting activity of pancreatic β cells to the epithelial cell proliferation promoting activity is twice or more that of betacellulin. The betacellulin mutein or a salt thereof according to claim 1, wherein 1 to 40 amino acid residues are deleted from the N-terminus. (1) an amino acid sequence represented by SEQ ID NO: 1, (2) an amino acid sequence in which 1 to 40 amino acids have been deleted from the N-terminal of the amino acid sequence represented by SEQ ID NO: 1, (3) SEQ ID NO: amino acid sequence or represented by 2 (4) SEQ ID nO: betacellulin mutein or a salt thereof 1 from the N-terminus of the amino acid sequence represented by 2 to claim 1, wherein of 40 amino acids deleted amino acid sequence . (1) SEQ ID NO: amino acid sequence or represented by 3 (2) SEQ ID NO: betacellulin mutein or salt thereof according to claim 1, wherein comprising the amino acid sequence represented by 4. SEQ ID NO: betacellulin mutein or salt thereof according to claim 1, wherein comprising the amino acid sequence represented by 37. SEQ ID NO: consisting of the amino acid sequence represented by 38 claim 1 betacellulin mutein or salt thereof according. A betacellulin mutein consisting of the amino acid sequence represented by SEQ ID NO: 44 or a salt thereof, wherein the differentiation promoting activity of pancreatic β cells is maintained and the epithelial cell proliferation promoting activity is reduced . A betacellulin mutein consisting of the amino acid sequence represented by SEQ ID NO: 45 or a salt thereof, wherein the differentiation promoting activity of pancreatic β cells is maintained and the epithelial cell proliferation promoting activity is reduced . 10. A transformant transformed with a recombinant vector containing a DNA encoding the betacellulin mutein according to any one of claims 1 to 9 is cultured to produce the betacellulin mutein. betacellulin muteins or preparation of a salt thereof according to any one of 1 to 9. A pharmaceutical composition comprising the betacellulin mutein or a salt thereof according to any one of claims 1 to 9 . 12. The pharmaceutical composition according to claim 11 , wherein the composition is a diabetes preventive / therapeutic agent.

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