JP2022509979A - 洗剤を混入した乾燥ハイドロゲル粒子および高分子の濃縮と比活性の向上 - Google Patents
洗剤を混入した乾燥ハイドロゲル粒子および高分子の濃縮と比活性の向上 Download PDFInfo
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- JP2022509979A JP2022509979A JP2021530303A JP2021530303A JP2022509979A JP 2022509979 A JP2022509979 A JP 2022509979A JP 2021530303 A JP2021530303 A JP 2021530303A JP 2021530303 A JP2021530303 A JP 2021530303A JP 2022509979 A JP2022509979 A JP 2022509979A
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Abstract
Description
ハイドロゲル粒子の種類:調製の難易度、調製コストおよび標的高分子との相互作用に応じて、ハイドロゲル粒子の種類を選択する。ポリアクリルアミドとポリデキストランの2種類のハイドロゲル粒子は、調製が比較的容易で経済的であること、および高分子と相互作用しない不活性であることから好ましく選択され、ポリアクリル酸またはポリアクリル酸塩のハイドロゲル粒子は、調製が容易で経済的であることに加え、ゲル質量あたりの吸水量が大きいことから好ましいが、この場合、帯びる負の電荷と標的高分子との相互作用が濃縮プロセスや回収率に及ぼす影響を慎重に検討する必要がある。
混入する洗剤の種類と濃度:標的高分子とハイドロゲル粒子の相互作用の特性に応じて、最高の質量および活性機能回収率が得られるような混入する洗剤の種類を選択し、洗剤混入液の濃度範囲は0.001~10%である。多糖や核酸以外のタンパク質などの敏感な生体高分子、特に活性機能が立体配座に非常に敏感な酵素には、好ましくはポリソルベート系、Brij系、NP-40、トリトン系などのマイルドな非イオン性混入する洗剤を使用し、ポリソルベート系は、ほとんどの生体高分子の活性機能に悪影響を及ぼさず、また、これらの高分子の活性機能の喪失を引き起こす凝集をしばしば防ぐことができるので、特に好ましい。
成分:アクリルアミド29g、N,N-メチレンビスアクリルアミド1g、脱イオン水100ml、過硫酸アンモニウム0.2g、テトラメチルエチレンジアミン40μl。
水相の調製:29gのアクリルアミド、1gのN,N-メチレンビスアクリルアミド、100mlの脱イオン水および0.2gの過硫酸アンモニウムを混合する。油相の調製:200mlのシクロヘキサンと3mlのスパン-80を均一に混合する。
図4に示すように、ステップ1では、実施例2または3で調製された洗剤を混入した乾燥ポリアクリルアミドハイドロゲル粒子を2g計量し、孔径20ミクロンのろ過膜と上部開口部に突出したキャッチを備えたプラスチックチューブに入れ、さらに、そのプラスチックチューブに、1mlあたり10~500μgの濃度でウシ血清アルブミン(図中の黒点)のpH7.4リン酸緩衝液を約12ml入れ、ステップ2では、ハイドロゲルとタンパク質溶液の混合体を5分間静置して、十分に水和・膨潤させ、ステップ3では、上記のプラスチックチューブを50mlの遠心分離管に嵌入し、3000 x gで3分間、水平回転ヘッドの細い矢印で示される円周方向に遠心分離し、ステップ4では、遠心分離スリーブの太い矢印で示される遠心力の方向に、約2mlのウシ血清アルブミン濃縮液を収集する。測定により、濃縮液タンパク質の質量回収率は90%以上であり、濃度は初期値の約6倍増加した。
実施例4で得られたウシ血清アルブミン濃縮液に10mlの脱イオン水を加え、さらに実施例2または3で調製された洗剤を混入した乾燥ポリアクリルアミドハイドロゲル粒子を2g入れたろ過膜プラスチックチューブに入れ、実施例4に記載の静置水和と遠心分離作業を繰り返して2mlの濃縮液を得る。この濃縮液は、実施例4で得られた濃縮液と比較して、塩およびリン酸の緩衝液の強度を約83%低減する。上記の脱塩および緩衝液の交換作業を1回繰り返すと、脱塩および緩衝液の交換率はさらに約97%まで向上する。
ウシ血清アルブミン試料を1mlあたり20~200μgの濃度のウシ小腸アルカリホスファターゼのPH7.5トリスヒドロキシメチルアミノメタン緩衝液に置き換えた以外は、実施例4と同様に12mlの試料量の濃縮作業を行う。得られた濃縮液の定量的タンパク質測定による質量回収率は90%であるが、アルカリホスファターゼ活性測定による活性回収率は100%であるため、アルカリホスファターゼの比活性が約11%向上する。
ヒトの尿に含まれる微量のタンパク質は、濃縮することでその濃度と対応する検出性を高めることができる。正常なヒトの尿に1mlあたり2μgの西洋ワサビペルオキシダーゼを混合したものをモデルタンパク尿試料とする。ウシ血清アルブミン試料を当該モデルタンパク尿試料に置き換えた以外は、実施例4と同様に12mlの試料量の濃縮作業を行う。最終的に得られた濃縮液に対して、西洋ワサビペルオキシダーゼ活性の測定を行ったところ、当該酵素タンパク質が約6倍濃縮され、回収率は95%以上であることがわかった。
配列決定やファージ展示のためにターゲットファージを選別する必要があるため、大腸菌内で組み入れられた後に細胞外培地に分泌されるM13ファージを濃縮する必要がある。ポリエチレングリコール沈殿に基づく既存のM13濃縮法は、大量の培地または低力価での回収率が低く、低温で長時間の作業も必要である。改良方法として、ウシ血清アルブミン試料を培地に分泌されるM13ファージ試料に置き換えた以外は、本発明の実施例4と同様に12mlの試料量の濃縮作業を行う。最終的な力価測定の結果、M13ファージは約6倍濃縮され、回収率は約95%であった。
Claims (9)
- 粒子の架橋ネットワークの孔径は、高分子またはタンパク質のサイズよりも小さいことを特徴とする、高分子の質量濃縮またはタンパク質の比活性向上のための洗剤を混入した乾燥ハイドロゲル粒子。
- 前記粒子は、ポリアクリルアミド、ポリアクリル酸、ポリアクリル酸塩、ポリデキストラン、セルロース、ポリビニルアルコール、ポリエチレングリコール、アガロース、ポリヒアルロン酸、ポリキチン、ポリアルギン酸、ポリアルギン酸ナトリウム、ポリビニルピロリドン、ポリペプチド、ポリプロテイン、上記ハイドロゲルモノマーの誘導体を重合して架橋した修飾ハイドロゲル、複数の上記ハイドロゲルモノマーまたはポリマーの混合物を重合して架橋したハイブリッドハイドロゲルから選択されることを特徴とする、請求項1に記載の粒子。
- 前記洗剤は、ポリソルベート-20、ポリソルベート-40、ポリソルベート-60、ポリソルベート-80、Brij-35、Brij-58、NP-40、トリトンX-100、トリトンX-114、オクチルグルコシド、オクチルチオグルコシド、3-[3-(コラミドプロピル)ジメチルアミノ]プロパンスルホン酸内塩、3-[(3-コラミドプロピル)ジメチルアミノ]-2-ヒドロキシ-1-プロパンスルホン酸内塩、コール酸ナトリウム、デオキシコール酸ナトリウム、セチルトリメチルアンモニウムブロマイド、ドデシル硫酸ナトリウムから選択されることを特徴とする、請求項1に記載の粒子。
- 前記洗剤の混入濃度は0.001~10%であることを特徴とする、請求項1に記載の粒子。
- 前記高分子液体試料の体積と請求項1~4に記載の粒子の質量を一定の比例で接触させ、前記粒子を膨潤させかつ前記高分子を排除し、前記高分子液体試料と前記粒子の混合体をろ過して遠心分離し、表面的に脱水したが内部の細孔が膨潤している前記粒子を除去し、前記粒子によって排除された前記高分子の濃縮ろ液を収集することを特徴とする、高分子質量濃縮の方法。
- 前記ろ過遠心分離は円錐形のろ過膜ドラムで行われ、ここで、前記高分子液体試料と前記粒子の混合体を小口径の底部に連続的に供給し、表面的に脱水したが内部の細孔が膨潤している前記粒子を遠心力によって大口径方向に向かって連続的に上方へ移動させて振り出し、前記高分子の濃縮ろ液を前記ろ過膜から連続的に振り出して収集することを特徴とする、請求項5に記載の方法。
- 前記高分子液体試料の接触膜によって隔離された余分な前記粒子は、定量的かつ制御可能に液体を吸収することを特徴とする、請求項5に記載の方法。
- 前記タンパク質液体試料の体積と請求項1~4に記載の粒子の質量を一定の比例で接触させ、前記粒子を膨潤させかつ前記タンパク質を排除し、そして、前記タンパク質液体試料と前記粒子の混合体をろ過して遠心分離し、表面的に脱水したが内部の細孔が膨潤している前記粒子を除去し、前記粒子によって排除された前記タンパク質の濃縮ろ液を収集することを特徴とする、タンパク質の比活性の向上方法。
- 前記タンパク質液体試料の体積と請求項1~4に記載の粒子の質量を一定の比例で接触させ、前記粒子を膨潤させかつ前記タンパク質を排除し、そして、前記タンパク質液体試料と前記粒子の混合体をろ過して遠心分離し、表面的に脱水したが内部の細孔が膨潤している前記粒子を除去し、前記粒子によって排除された前記タンパク質の濃縮ろ液を収集することを特徴とする、結晶化または立体配座分析のためのタンパク質液体試料を処理する方法。
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