JPWO2015034056A1 - アフィニティー分離マトリックス用分離能強化リガンド - Google Patents
アフィニティー分離マトリックス用分離能強化リガンド Download PDFInfo
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Abstract
Description
(1)配列番号1のアミノ酸配列、または配列番号2のアミノ酸配列において、第7位、第18位、第39位、第40位、第46位、第54位、および、第55位のアミノ酸からなる群から選択される1以上のアミノ酸が、酸性アミノ酸に置換されているアミノ酸配列を有する免疫グロブリンG結合性ペプチド;
(2)上記(1)のアミノ酸配列において、さらに第7位、第18位、第39位、第40位、第46位、第54位、および、第55位のアミノ酸以外の、1または数個のアミノ酸が置換、挿入、欠失および/または付加されているアミノ酸配列を有する免疫グロブリンG結合性ペプチド;
(3)上記(1)のアミノ酸配列に対して80%以上の配列同一性を有するアミノ酸配列を有する免疫グロブリンG結合性ペプチド(但し、上記(1)において置換により導入された酸性アミノ酸は維持されるものとする)
に関する。
(1)配列番号1のアミノ酸配列、または配列番号2のアミノ酸配列において、第7位、第18位、第39位、第40位、第46位、第54位、および、第55位のアミノ酸からなる群から選択される1以上のアミノ酸が、酸性アミノ酸に置換されているアミノ酸配列を有する免疫グロブリンG結合性ペプチド;
(2)上記(1)のアミノ酸配列において、さらに第7位、第18位、第39位、第40位、第46位、第54位、および、第55位のアミノ酸以外の、1または数個のアミノ酸が置換、挿入、欠失および/または付加されているアミノ酸配列を有する免疫グロブリンG結合性ペプチド;
(3)上記(1)のアミノ酸配列に対して80%以上の配列同一性を有するアミノ酸配列を有する免疫グロブリンG結合性ペプチド(但し、上記(1)において置換により導入された酸性アミノ酸は維持されるものとする)。
酸性アミノ酸を導入する部位の評価には、Zドメイン(Z−Wild.1d、配列番号2)の立体構造を利用した。タンパク質立体構造データベースPDB(http://www.rcsb.org/pdb/home/home.do)にある公知のZドメインの立体構造(PDBコード:2SPZ)を用いて、各アミノ酸残基の溶媒露出面積(ASA)をDSSPを用いて算出した。アミノ酸側鎖の大きさの違いによる影響を補正するため、rASA値(ASA(Xxx)/ASA(Gly−Xxx−Gly)、Xxxは計算対象の任意のアミノ酸)も算出した。この各残基のASA値/rASA値を表1に示す。
プロテインAのCドメインに、実施例1で設計した酸性アミノ酸への置換変異を導入して得られるペプチドを、大腸菌発現系を利用して取得した。
(0)C−G29A/S33E.1d(配列番号3、比較例として)
(1)C−G29A/S33E/Q55E.1d(配列番号4)
(2)C−G29A/S33E/K7E/A46E.1d(配列番号5)
(3)C−G29A/S33E/K7E/H18D/V40D/A46D/Q55E.1d(配列番号6)
表面プラズモン共鳴を利用したバイオセンサーBiacore 3000(GEヘルスケア・バイオサイエンス社)を用いて、実施例2で取得したペプチドの免疫グロブリンとの親和性を解析した。ヒト血漿から分画したヒト免疫グロブリンG製剤(以後は、ヒトIgGと記する)をセンサーチップに固定化し、各ペプチドをチップ上に流して、両者の相互作用を検出した。
実施例2で取得した各種単ドメイン型Cドメイン変異体の陰イオン交換クロマトグラフィー実験を行い、変異体の陰イオン性の違いを解析した。
Claims (22)
- 下記(1)〜(3)のいずれかの免疫グロブリンG結合性ペプチド:
(1)配列番号1のアミノ酸配列、または配列番号2のアミノ酸配列において、第7位、第18位、第39位、第40位、第46位、第54位、および、第55位のアミノ酸からなる群から選択される1以上のアミノ酸が、酸性アミノ酸に置換されているアミノ酸配列を有する免疫グロブリンG結合性ペプチド;
(2)上記(1)のアミノ酸配列において、さらに第7位、第18位、第39位、第40位、第46位、第54位、および、第55位のアミノ酸以外の、1または数個のアミノ酸が置換、挿入、欠失および/または付加されているアミノ酸配列を有する免疫グロブリンG結合性ペプチド;
(3)上記(1)のアミノ酸配列に対して80%以上の配列同一性を有するアミノ酸配列を有する免疫グロブリンG結合性ペプチド(但し、上記(1)において置換により導入された酸性アミノ酸は維持されるものとする)。 - 前記酸性アミノ酸が、側鎖にカルボキシル基を有する酸性アミノ酸である、請求項1に記載の免疫グロブリンG結合性ペプチド。
- 前記側鎖にカルボキシル基を有する酸性アミノ酸が、アスパラギン酸またはグルタミン酸である、請求項2に記載の免疫グロブリンG結合性ペプチド。
- 前記酸性アミノ酸に置換されるアミノ酸が、第7位、第18位、第40位、第46位、および、第55位のアミノ酸からなる群から選択される1以上のアミノ酸である、請求項1〜3のいずれかに記載の免疫グロブリンG結合性ペプチド。
- 前記置換、挿入、欠失および/または付加されるアミノ酸が、第1位、第2位、第3位、第4位、第6位、第23位、第29位、第33位、第35位、第36位、第37位、第42位、第49位、第50位、第57位、および、第58位のアミノ酸からなる群から選択される1以上のアミノ酸である、請求項1〜4のいずれかに記載の免疫グロブリンG結合性ペプチド。
- 前記置換、挿入、欠失および/または付加されるアミノ酸が、第29位、第33位、第36位、および、第37位のアミノ酸からなる群から選択される1以上のアミノ酸である、請求項5に記載の免疫グロブリンG結合性ペプチド。
- 2以上のアミノ酸が酸性アミノ酸に置換されている、請求項1〜6のいずれかに記載の免疫グロブリンG結合性ペプチド。
- 3以上のアミノ酸が酸性アミノ酸に置換されている、請求項1〜7のいずれかに記載の免疫グロブリンG結合性ペプチド。
- 4以上のアミノ酸が酸性アミノ酸に置換されている、請求項1〜8のいずれかに記載の免疫グロブリンG結合性ペプチド。
- 請求項1〜9のいずれかに記載の免疫グロブリンG結合性ペプチドを2個以上連結した複数ドメインを有する、免疫グロブリンG結合性ペプチド。
- 請求項1〜10のいずれかに記載の免疫グロブリンG結合性ペプチドをコードする塩基配列を含むDNA。
- 請求項11に記載のDNAを含むベクター。
- 請求項12に記載のベクターを宿主細胞に導入して得られる形質転換体。
- 請求項1〜10のいずれかに記載の免疫グロブリンG結合性ペプチドを水不溶性担体に固定化して得られるアフィニティー分離マトリックス。
- 免疫グロブリンGまたは免疫グロブリンG誘導体を請求項14に記載のアフィニティー分離マトリックスと接触させる工程、および、アフィニティー分離マトリックスに結合した免疫グロブリンGまたは免疫グロブリンG誘導体を、アフィニティー分離マトリックスから分離する工程を含む、免疫グロブリンGまたは免疫グロブリンG誘導体の製造方法。
- アフィニティー精製用リガンドのアミノ酸配列中の酸性アミノ酸数を増加させることにより、陽イオン交換能を向上させる工程を含む、改良型アフィニティー精製用リガンドの製造方法。
- 酸性アミノ酸数の増加が、酸性アミノ酸への置換変異、および/または酸性アミノ酸の付加によるものである、請求項16に記載の改良型アフィニティー精製用リガンドの製造方法。
- 前記アフィニティー精製の対象が、免疫グロブリンGまたは免疫グロブリンG誘導体である、請求項16または17に記載の改良型アフィニティー精製用リガンドの製造方法。
- 前記アフィニティー精製用リガンドが、プロテインA、プロテインG、または、プロテインLの免疫グロブリンG結合性ドメインである、請求項16〜18のいずれかに記載の改良型アフィニティー精製用リガンドの製造方法。
- 前記酸性アミノ酸数の増加が、2以上の増加である、請求項16〜19のいずれかに記載の改良型アフィニティー精製用リガンドの製造方法。
- 前記酸性アミノ酸数の増加が、3以上の増加である、請求項16〜20のいずれかに記載の改良型アフィニティー精製用リガンドの製造方法。
- 前記酸性アミノ酸数の増加が、4以上の増加である、請求項16〜21のいずれかに記載の改良型アフィニティー精製用リガンドの製造方法。
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WO2016167143A1 (ja) * | 2015-04-13 | 2016-10-20 | 富士フイルム株式会社 | 短鎖ペプチド固定化担体の製造方法および短鎖ペプチド固定化担体 |
JP6891114B2 (ja) * | 2015-07-22 | 2021-06-18 | 株式会社カネカ | 酸性pH領域での抗体結合能が低下した抗体結合性タンパク質 |
WO2017022759A1 (ja) * | 2015-08-04 | 2017-02-09 | 株式会社カネカ | 免疫グロブリン結合性改変型タンパク質 |
CN116731197A (zh) * | 2016-09-19 | 2023-09-12 | 豪夫迈·罗氏有限公司 | 基于补体因子的亲和层析 |
US20210179671A1 (en) * | 2018-08-24 | 2021-06-17 | Jsr Corporation | Immunoglobulin-binding protein, and affinity carrier using same |
GB202113626D0 (en) | 2021-09-24 | 2021-11-10 | Cytiva Bioprocess R & D Ab | FC binding polypeptides |
GB202203478D0 (en) | 2022-03-14 | 2022-04-27 | Cytiva Bioprocess R & D Ab | Vh3 binding polypeptides |
CN115820704B (zh) * | 2022-12-22 | 2023-06-16 | 武汉爱博泰克生物科技有限公司 | 表达蛋白l的重组质粒及其应用与重组蛋白l的表达方法 |
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EP0326046A3 (en) | 1988-01-25 | 1990-06-13 | Takeda Chemical Industries, Ltd. | Production of human epidermal growth factor |
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JP2006304633A (ja) * | 2005-04-26 | 2006-11-09 | Apro Life Science Institute Inc | イムノグロブリン結合タンパク質 |
WO2008044692A1 (fr) * | 2006-10-10 | 2008-04-17 | National Institute Of Advanced Industrial Science And Technology | Protéine adaptée pour une commande d'orientation/immobilisation de protéine et support d'immobilisation destiné à la protéine |
WO2012083425A1 (en) * | 2010-12-21 | 2012-06-28 | The University Of Western Ontario | Novel alkali-resistant variants of protein a and their use in affinity chromatography |
WO2012133349A1 (ja) * | 2011-03-25 | 2012-10-04 | 株式会社カネカ | アフィニティー分離マトリックス用タンパク質 |
JP2015511637A (ja) * | 2012-03-28 | 2015-04-20 | ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ | アフィニティークロマトグラフィーマトリックス |
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