JP2004189657A - PEPTIDE OR PROTEIN HAVING BINDABILITY TO Fc FRAGMENT OF IgG - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel peptide or protein having bindability to the Fc fragment of IgG and being utilizable for e.g., the purification or detection of IgG. <P>SOLUTION: The Fc fragment of IgG of an animal origin is used as a target substance. The amino acid sequence of a peptide having bindability to the target substance is identified by screening by a pharge display method. A peptide obtained in this way and having an amino acid sequence represented by sequence numbers 1 to 164 or having an amino acid sequence obtained by subjecting to modification or the substitution, insertion, and/or elimination of amino acid residues within the scope not detrimental to the bindability to the Fc fragment of IgG in the amino acid sequence is advantageous in that it can be chemically synthesized and can be prepared in a large quantity at a low cost. Further, it has no risk of e.g. contamination with foreign matter and therefore can be desirably used for e.g. the purification of an antibody especially for medical use. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００７２】
表１から、配列番号１６５〜２０７で表されるペプチドは、ｈｕｍａｎ Ｆｃに対する親和性が異なるものの、いずれもｈｕｍａｎ Ｆｃに対して結合力を有していることが分かる。
【００７３】
（４）得られたペプチドの共通配列の検索
配列番号１６５〜２０７で表されるペプチドのアミノ酸配列から、表２のような組み合わせで共通配列の検索を行い、配列番号１〜８７で表される共通配列を決定した。
【００７４】
【表２】

【００７５】
実施例２
実施例１のｈｕｍａｎ Ｆｃの代わりに、ヒト由来ＩｇＧ（ＣＡＰＰＥＬ社製）そのものを用い、対照としてブタ、ウシ、マウス、ヤギ、ウサギ由来の各種ＩｇＧ（ＲＯＣＫＬＡＮＤ社製）を用いた以外は、実施例１の（２）と同様の方法で実験を行い、配列番号１６５〜１６７、１６９、１７０、１７２、１７３、１７５〜１８９で表されるペプチドのヒト由来ＩｇＧに対する特異性を検討した。その結果を図２に示す。
【００７６】
図２から分かるように、実施例１の場合とほぼ同様の結果が得られ、配列番号１６５〜１６７、１６９、１７０、１７２、１７３、１７５〜１８９で表されるペプチドは、ヒト由来ＩｇＧそのものに対しても特異性が高いことが確認された。
【００７７】
実施例３
配列番号１６６で表されるペプチドのＣ末端側にグリシンとアルギニンを付加した配列番号２８１で表されるペプチドを用いて、ｈｕｍａｎ Ｆｃが精製できるかどうかを確認した。なお、Ｃ末端に付加したグリシンはスペーサーとして、アルギニンはアミノ基を利用したカラムへの固定化に利用するためのアミノ基追加用である。
【００７８】
配列番号２８１で表されるペプチドは、株式会社シグマジェノシスに合成を依頼（８０％純度保証）した。
【００７９】
＜カラムへの固定化＞
ｈｕｍａｎ Ｆｃを精製するために、配列番号２８１で表されるペプチドを固定化したカラムを、商品名「ＨｉＴｒａｐ ＮＨＳ−Ａｃｔｉｖａｔｅｄ ＨＰ（カラムボリューム１ｍｌ）」（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ社製、以下、単にカラムという）を用いて、以下のようにして作製した。
【００８０】
まず、カラムのキャップを外し、シリンジを用いて氷冷した６ｍｌの１ｍＭ ＨＣｌでカラムを洗浄した後、配列番号２８１で表されるペプチドを含む溶液（１ｍｇ／ｍｌ溶液、バッファ：炭酸バッファ０．２Ｍ ＮａＨＣＯ_３、０．５Ｍ ＮａＣｌ、ｐＨ８．３）１．５ｍｌを上記カラムにシリンジで注入し、これを室温で２時間放置してペプチドをカラムに固定化した。ペプチド固定化後、未反応のカラム官能基をブロックして活性を無くすため、エタノールアミン溶液（０．５Ｍエタノールアミン、０．５Ｍ ＮａＣｌ、ｐＨ８．３）６ｍｌと酢酸バッファ（０．１Ｍ酢酸、０．５Ｍ ＮａＣｌ、ｐＨ４．０）６ｍｌを交互に４回ずつ通した。なお、途中、２回目のエタノールアミン溶液注入後は３０分室温で放置した。この操作終了後、ＰＢＳ（１０ｍＭリン酸、１４０ｍＭ ＮａＣｌ、ｐＨ７．４）５ｍｌでカラムを平衡化した。
【００８１】
＜Ｐｕｒｉｆｉｃａｔｉｏｎ＞
上記のようにして作成したカラムを用い、以下のようにしてｈｕｍａｎ Ｆｃを精製した。なお、精製にはＨＰＬＣシステム「ＡＫＴＡ ｐｕｒｉｆｉｅｒ」（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ社製）を用いた。
【００８２】
まず、１００μｇ／ｍｌとなるようにｈｕｍａｎ Ｆｃ（ＩＣＮ／ＣＡＰＰＥＬ社製、ＰＵＲＩＦＩＥＤ ＨＵＭＡＮ ＩＧＧ ＦＣ）をＮａＨＣＯ_３溶液（１００ｍＭ、ｐＨ８．０）に溶解してｈｕｍａｎ Ｆｃ溶液を調製した。対照として、ヒトに近いと言われるブタ由来ＩｇＧ Ｆｃフラグメント（ＲＯＣＫＬＡＮＤ社製、ＳＷＩＮＥ ＩｇＧ Ｆｃ ｆｒａｇｍｅｎｔ）をした溶液も同様に調製した。そして、カラムをＨＰＬＣシステムに繋げ、１ｍｌ／ｍｉｎの流速でＰＢＳで平衡化した（実験を通して流速はこのままである。）。これに、上記ｈｕｍａｎ Ｆｃ溶液１００μｌを注入してカラムに吸着させた。そして、カラム容量の５倍のボリューム（５ｍｌ）のＰＢＳで洗浄した後、連続して溶離液（ＰＢＳ ｐＨ３．０）をグラディエント投入した。グラディエントは、溶離液１００％になるまでカラム容量の２０倍のボリューム（２０ｍｌ）と設定した。また、ブタ由来ＩｇＧ Ｆｃフラグメント溶液も上記と同様にしてカラムに供した。その溶出結果を図３に示す。
【００８３】
実施例４
カラムに固定化するペプチドとして、配列番号１７３で表されるペプチドのＣ末端側にグリシンとアルギニンを付加した配列番号２８２で表されるペプチドを用いた以外は、実施例３と同様の方法でカラムを作製し、ｈｕｍａｎ Ｆｃの精製を行った。その結果を図３に示す。なお、配列番号１７３で表されるペプチドは、実施例３で用いた配列番号１６６で表されるペプチドに比べて、ｈｕｍａｎ Ｆｃに対する親和性が弱いことが、ＥＬＩＳＡ発色により確認されている（表１参照）。
【００８４】
実施例５
カラムに固定化するペプチドとして、配列番号２８１で表されるペプチドと、配列番号２８２で表されるペプチドを用いた以外は、実施例３と同様の方法でカラムを作製し、ｈｕｍａｎ Ｆｃの精製を行った。なお、これらのペプチドをカラムに固定化する際には、それぞれ０．５ｍｇ／ｍｌ（あわせて１ｍｇ／ｍｌ）となるように炭酸バッファに溶解して固定化した。その結果を図３に示す。
【００８５】
図３中、（Ａ）はサンプルを負荷していないカラム（コントロール）の溶出結果、（Ｂ）は配列番号２８１で表されるペプチドを固定したカラムにｈｕｍａｎ Ｆｃを負荷した場合の溶出結果（実施例３）、（Ｃ）は配列番号２８１で表されるペプチドを固定したカラムにブタ由来ＩｇＧ Ｆｃフラグメントを負荷した場合の溶出結果（実施例３）、（Ｄ）は配列番号２８２で表されるペプチドを固定したカラムにｈｕｍａｎ Ｆｃを負荷した場合の溶出結果（実施例４）、（Ｅ）は、配列番号２８１で表されるペプチド及び配列番号２８２で表されるペプチドを固定したカラムにｈｕｍａｎ Ｆｃを負荷した場合の溶出結果（実施例５）を表し、▲１▼〜▲７▼は検出されたピークを示す。
【００８６】
図３の（Ｂ）、（Ｃ）の結果から、配列番号２８１で表されるペプチドを固定したカラムにはｈｕｍａｎ Ｆｃは吸着されているが、ブタ由来ＩｇＧ Ｆｃフラグメントは吸着されていないことが分かる。したがって、ｈｕｍａｎ Ｆｃに結合性を有するペプチドを用いることにより、ｈｕｍａｎ Ｆｃを特異的に精製できることが分かる。なお、（Ｂ）において、複数ピーク（▲１▼〜▲３▼）が見られるのは、ＩｇＧのサブクラスＩｇＧ１〜ＩｇＧ４による結合親和性の違いから来るものと考えられ、サブクラスを分けるカラムも作成可能であることが示唆される。
【００８７】
一方、図３の（Ｄ）の結果から、配列番号２８２で表されるペプチドを固定したカラムを用いた場合、実施例３のように、溶離液を用いなくてもｈｕｍａｎ Ｆｃが溶出されていることが分かる。したがって、ｈｕｍａｎ Ｆｃに対する親和性の弱いペプチドを用いることにより、リテンションタイムの違いによってＩｇＧの精製が可能であることが示され、ＩｇＧを精製する際に問題となるＩｇＧの失活や変性を防止できる可能性が示唆される。
【００８８】
また、図３の（Ｅ）の結果から、配列番号２８１で表されるペプチド及び配列番号２８２で表されるペプチドを固定したカラムを用いてもｈｕｍａｎ Ｆｃを十分に精製できることが分かる。なお、ピーク（▲６▼、▲７▼）の状態が上記２つのペプチドをそれぞれ単独で用いた場合（実施例３、４の場合）と異なることから、ペプチドを適当に組み合わせることにより、目的に合ったカラムが作製できる可能性も示唆される。
【００８９】
実施例６
ターゲット物質として、ウマ由来ＩｇＧのＦｃフラグメント（以下、ｈｏｒｓｅ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｈｏｒｓｅ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２０８〜２１２で表されるペプチドのアミノ酸配列を同定した。
【００９０】
得られた全てのペプチド（配列番号２０８〜２１２）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｈｏｒｓｅ Ｆｃに対する結合性を調べた。その結果を表３に示す。なお、表３中の「発色値」、ｈｏｒｓｅ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｈｏｒｓｅ Ｆｃに対する親和性が強いことを示す。
【００９１】
【表３】

【００９２】
表３から、配列番号２０８〜２１２で表されるペプチドは、ｈｏｒｓｅ Ｆｃに対する親和性が異なるものの、いずれもｈｏｒｓｅ Ｆｃに対して結合力を有していることが分かる。
【００９３】
また、配列番号２０８〜２１２で表されるペプチドのアミノ酸配列から、表４のような組み合わせで共通配列の検索を行い、配列番号８８〜９０で表される共通配列を決定した。
【００９４】
【表４】

【００９５】
実施例７
ターゲット物質として、ヒツジ由来ＩｇＧのＦｃフラグメント（以下、ｓｈｅｅｐ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｓｈｅｅｐ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２１３〜２１６で表されるペプチドのアミノ酸配列を同定した。
【００９６】
得られた全てのペプチド（配列番号２１３〜２１６）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｓｈｅｅｐ Ｆｃに対する結合性を調べた。その結果を表５に示す。なお、表５中の「発色値」は、ｓｈｅｅｐ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｓｈｅｅｐ Ｆｃに対する親和性が強いことを示す。
【００９７】
【表５】

【００９８】
表５から、配列番号２１３〜２１６で表されるペプチドは、ｓｈｅｅｐ Ｆｃに対する親和性が異なるものの、いずれもｓｈｅｅｐ Ｆｃに対して結合力を有していることが分かる。
【００９９】
また、配列番号２１３〜２１６で表されるペプチドのアミノ酸配列から、表６のような組み合わせで共通配列の検索を行い、配列番号９１〜９３で表される共通配列を決定した。
【０１００】
【表６】

【０１０１】
実施例８
ターゲット物質として、ウサギ由来ＩｇＧのＦｃフラグメント（以下、ｒａｂｂｉｔ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｒａｂｂｉｔ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２１７〜２２０で表されるペプチドのアミノ酸配列を同定した。
【０１０２】
得られた全てのペプチド（配列番号２１７〜２２０）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｒａｂｂｉｔ Ｆｃに対する結合性を調べた。その結果を表７に示す。なお、表７中の「発色値」は、ｒａｂｂｉｔ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｒａｂｂｉｔ Ｆｃに対する親和性が強いことを示す。
【０１０３】
【表７】

【０１０４】
表７から、配列番号２１７〜２２０で表されるペプチドは、ｒａｂｂｉｔ Ｆｃに対する親和性が異なるものの、いずれもｒａｂｂｉｔ Ｆｃに対して結合力を有していることが分かる。
【０１０５】
また、配列番号２１７〜２２０で表されるペプチドのアミノ酸配列から、表８のような組み合わせで共通配列の検索を行い、配列番号９４〜９７で表される共通配列を決定した。
【０１０６】
【表８】

【０１０７】
実施例９
ターゲット物質として、モルモット由来ＩｇＧのＦｃフラグメント（以下、ｇｕｉｎｅａ ｐｉｇ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｇｕｉｎｅａ ｐｉｇ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２２１〜２２７で表されるペプチドのアミノ酸配列を同定した。
【０１０８】
得られた全てのペプチド（配列番号２２１〜２２７）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｇｕｉｎｅａ ｐｉｇ Ｆｃに対する結合性を調べた。その結果を表９に示す。なお、表９中の「発色値」は、ｇｕｉｎｅａ ｐｉｇ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｇｕｉｎｅａ ｐｉｇ Ｆｃに対する親和性が強いことを示す。
【０１０９】
【表９】

【０１１０】
表９から、配列番号２２１〜２２７で表されるペプチドは、ｇｕｉｎｅａ ｐｉｇ Ｆｃに対する親和性が異なるものの、いずれもｇｕｉｎｅａ ｐｉｇ Ｆｃに対して結合力を有していることが分かる。
【０１１１】
また、配列番号２２１〜２２７で表されるペプチドのアミノ酸配列から、表１０のような組み合わせで共通配列の検索を行い、配列番号９８〜１０４で表される共通配列を決定した。
【０１１２】
【表１０】

【０１１３】
実施例１０
ターゲット物質として、ヤギ由来ＩｇＧのＦｃフラグメント（以下、ｇｏａｔ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｇｏａｔ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２２８〜２３２で表されるアミノ酸配列を同定した。
【０１１４】
得られた全てのペプチド（配列番号２２８〜２３２）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｇｏａｔ Ｆｃに対する結合性を調べた。その結果を表１１に示す。なお、表１１中の「発色値」は、ｇｏａｔ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｇｏａｔ Ｆｃに対する親和性が強いことを示す。
【０１１５】
【表１１】

【０１１６】
表１１から、配列番号２２８〜２３２で表されるペプチドは、ｇｏａｔ Ｆｃに対する親和性が異なるものの、いずれもｇｏａｔ Ｆｃに対して結合力を有していることが分かる。
【０１１７】
また、配列番号２２８〜２３２で表されるペプチドのアミノ酸配列から、表１２のような組み合わせで共通配列の検索を行い、配列番号１０５〜１０８で表される共通配列を決定した。
【０１１８】
【表１２】

【０１１９】
実施例１１
ターゲット物質として、ネコ由来ＩｇＧのＦｃフラグメント（以下、ｃａｔ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｃａｔ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２３３〜２４８で表されるペプチドのアミノ酸配列を同定した。
【０１２０】
得られた全てのペプチド（配列番号２３３〜２４８）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｃａｔ Ｆｃに対する結合性を調べた。その結果を表１３に示す。なお、表１３中の「発色値」は、ｃａｔ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｃａｔ Ｆｃに対する親和性が強いことを示す。
【０１２１】
【表１３】

【０１２２】
表１３から、配列番号２３３〜２４８で表されるペプチドは、ｃａｔ Ｆｃに対する親和性が異なるものの、いずれもｃａｔ Ｆｃに対して結合力を有していることが分かる。
【０１２３】
また、配列番号２３３〜２４８で表されるペプチドのアミノ酸配列から、表１４のような組み合わせで共通配列の検索を行い、配列番号１０９〜１３１で表される共通配列を決定した。
【０１２４】
【表１４】

【０１２５】
実施例１２
ターゲット物質として、イヌ由来ＩｇＧのＦｃフラグメント（以下、ｄｏｇ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｄｏｇ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２４９〜２６１で表されるペプチドのアミノ酸配列を同定した。
【０１２６】
得られた全てのペプチド（配列番号２４９〜２６１）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｄｏｇ Ｆｃに対する結合性を調べた。その結果を表１５に示す。なお、表１５中の「発色値」は、ｄｏｇ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｄｏｇ Ｆｃに対する親和性が強いことを示す。
【０１２７】
【表１５】

【０１２８】
表１５から、配列番号２４９〜２６１で表されるペプチドは、ｄｏｇ Ｆｃに対する親和性が異なるものの、いずれもｄｏｇ Ｆｃに対して結合力を有していることが分かる。
【０１２９】
また、配列番号２４９〜２６１で表されるペプチドのアミノ酸配列から、表１６のような組み合わせで共通配列の検索を行い、配列番号１３２〜１５１で表される共通配列を決定した。
【０１３０】
【表１６】

【０１３１】
実施例１３
ターゲット物質として、ウシ由来ＩｇＧのＦｃフラグメント（以下、ｂｏｖｉｎｅ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｂｏｖｉｎｅ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２６２〜２６７で表されるペプチドのアミノ酸配列を同定した。
【０１３２】
得られた全てのペプチド（配列番号２６２〜２６７）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｂｏｖｉｎｅ Ｆｃに対する結合性を調べた。その結果を表１７に示す。なお、表１７中の「発色値」は、ｂｏｖｉｎｅ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｂｏｖｉｎｅ Ｆｃに対する親和性が強いことを示す。
【０１３３】
【表１７】

【０１３４】
表１７から、配列番号２６２〜２６７で表されるペプチドは、ｂｏｖｉｎｅ Ｆｃに対する親和性が異なるものの、いずれもｂｏｖｉｎｅ Ｆｃに対して結合力を有していることが分かる。
【０１３５】
また、配列番号２６２〜２６７で表されるペプチドのアミノ酸配列から、表１８のような組み合わせで共通配列の検索を行い、配列番号１５２〜１５６で表される共通配列を決定した。
【０１３６】
【表１８】

【０１３７】
実施例１４
ターゲット物質として、ブタ由来ＩｇＧのＦｃフラグメント（以下、ｓｗｉｎｅ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｓｗｉｎｅ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２６８〜２７７で表されるペプチドのアミノ酸配列を同定した。
【０１３８】
得られた全てのペプチド（配列番号２６８〜２７７）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｓｗｉｎｅ Ｆｃに対する結合性を調べた。その結果を表１９に示す。なお、表１９中の「発色値」は、ｓｗｉｎｅ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｓｗｉｎｅ Ｆｃに対する親和性が強いことを示す。
【０１３９】
【表１９】

【０１４０】
表１９から、配列番号２６８〜２７７で表されるペプチドは、ｓｗｉｎｅ Ｆｃに対する親和性が異なるものの、いずれもｓｗｉｎｅ Ｆｃに対して結合力を有していることが分かる。
【０１４１】
また、配列番号２６８〜２７７で表されるペプチドのアミノ酸配列から、表２０のような組み合わせで共通配列の検索を行い、配列番号１５７〜１６２で表される共通配列を決定した。
【０１４２】
【表２０】

【０１４３】
実施例１５
ターゲット物質として、マウス由来ＩｇＧのＦｃフラグメント（以下、ｍｏｕｓｅ Ｆｃと略記する。）を用いた以外は、実施例１の（１）と同様にしてファージディスプレイ法（選択操作４回）を行い、ｍｏｕｓｅ Ｆｃに対して結合性を有するペプチドのスクリーニングを行い、配列番号２７８〜２８０で表されるペプチドのアミノ酸配列を同定した。
【０１４４】
得られた全てのペプチド（配列番号２７８〜２８０）について、実施例１（２）と同様の方法（ＥＬＩＳＡ法）で、ｍｏｕｓｅ Ｆｃに対する結合性を調べた。その結果を表２１に示す。なお、表２１中の「発色値」は、ｍｏｕｓｅ Ｆｃ（ターゲット物質）固定化ウェルの吸光度（４０５ｎｍ）／コントロール（空）ウェルの吸光度（４０５ｎｍ）を表し、「発色値」が大きいほど、ｍｏｕｓｅ Ｆｃに対する親和性が強いことを示す。
【０１４５】
【表２１】

【０１４６】
表２１から、配列番号２７８〜２８０で表されるペプチドは、ｍｏｕｓｅ Ｆｃに対する親和性が異なるものの、いずれもｍｏｕｓｅ Ｆｃに対して結合力を有していることが分かる。
【０１４７】
また、配列番号２７８〜２８０で表されるペプチドのアミノ酸配列から、表２２のような組み合わせで共通配列の検索を行い、配列番号１６３〜１６４で表される共通配列を決定した。
【０１４８】
【表２２】

【０１４９】
「配列表フリーテキスト」
配列番号１〜８７：ファージディスプレイ法で得られたヒト由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号８８〜９０：ファージディスプレイ法で得られたウマ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号９１〜９３：ファージディスプレイ法で得られたヒツジ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号９４〜９７：ファージディスプレイ法で得られたウサギ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定されたアミノ酸配列である。
配列番号９８〜１０４：ファージディスプレイ法で得られたモルモット由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号１０５〜１０８：ファージディスプレイ法で得られたヤギ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号１０９〜１３１：ファージディスプレイ法で得られたネコ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号１３２〜１５１：ファージディスプレイ法で得られたイヌ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号１５２〜１５６：ファージディスプレイ法で得られたウシ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号１５７〜１６２：ファージディスプレイ法で得られたブタ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号１６３〜１６４：ファージディスプレイ法で得られたマウス由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドから決定された共通配列である。
配列番号１６５〜２０７：ファージディスプレイ法によって得られた、ヒト由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２０８〜２１２：ファージディスプレイ法によって得られた、ウマ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２１３〜２１６：ファージディスプレイ法によって得られた、ヒツジ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２１７〜２２０：ファージディスプレイ法によって得られた、ウサギ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２２１〜２２７：ファージディスプレイ法によって得られた、モルモット由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２２８〜２３２：ファージディスプレイ法によって得られた、ヤギ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２３３〜２４８：ファージディスプレイ法によって得られた、ネコ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２４９〜２６１：ファージディスプレイ法によって得られた、イヌ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２６２〜２６７：ファージディスプレイ法によって得られた、ウシ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２６８〜２７７：ファージディスプレイ法によって得られた、ブタ由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２７８〜２８０：ファージディスプレイ法によって得られた、マウス由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
配列番号２８１：配列番号１６６で表されるペプチドのＣ末端側にグリシンとアルギニンを付加したペプチドであって、ヒト由来ＩｇＧのＦｃフラグメントを精製する際のアフィニティリガンドとして用いたペプチドである。
配列番号２８２：配列番号１７３で表されるペプチドのＣ末端側にグリシンとアルギニンを付加したペプチドであって、ヒト由来ＩｇＧのＦｃフラグメントを精製する際のアフィニティリガンドとして用いたペプチドである。
配列番号２８３〜２８９：非特許文献１に記載されたヒト由来ＩｇＧのＦｃフラグメントに結合性を有するペプチドである。
【０１５０】
【発明の効果】
以上説明したように本発明によれば、ヒト、ウマ、ヒツジ、ウサギ、モルモット、ヤギ、ネコ、イヌ、ウシ、ブタ、マウス等のＩｇＧのＦｃフラグメントに結合性を有する新規なペプチド又はタンパク質をそれぞれ提供することができる。これらのペプチドは、各動物種由来のＩｇＧのＦｃフラグメントに対する特異性が高いので、各動物由来のＩｇＧの精製、検出、定性、ＩｇＧのＦｃ部分とＦｃレセプター間の結合阻害による研究試薬、医薬等に利用することができる。
【０１５１】
これらのペプチドは、化学合成可能であり、低コストで大量に調製することができるという利点がある。また、夾雑物の混入等の心配がないので、医薬用途の抗体の精製にも利用しやすいという利点もある。例えば、本発明で提供されるペプチドをＩｇＧ精製用のアフィニティリガンドとして用いた場合は、ＩｇＧのサブクラスを分けることが可能なカラムを作製できる可能性がある。また、ＩｇＧのＦｃフラグメントに対する親和性の弱いペプチドを用いることにより、リテンションタイムの違いでＩｇＧの精製が可能なカラムを作製できる可能性もあり、精製工程におけるＩｇＧの失活や変性を防止できる可能性がある。
【０１５２】
【配列表】

【図面の簡単な説明】
【図１】配列番号１６５〜１６７、１６９、１７０、１７２、１７３、１７５〜１８９で表されるペプチドのｈｕｍａｎ Ｆｃに対する結合性の確認試験の結果を表す図である。
【図２】配列番号１６５〜１６７、１６９、１７０、１７２、１７３、１７５〜１８９で表されるペプチドのヒト由来ＩｇＧに対する特異性を検討した結果を示す図である。
【図３】配列番号２８１、２８２で表されるペプチドを固定化したカラムを用いてｈｕｍａｎ Ｆｃを精製した際の溶出結果を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a peptide or protein having a binding property to an IgG Fc fragment which can be used for purification, detection and the like of IgG.
[0002]
[Prior art]
Antibodies, which play a central role in the immune response, have been used in a wide range of fields, including medicine and clinical diagnosis. In recent years, antibody-based drugs (antibody drugs) have been actively developed. Is being done.
[0003]
Antibodies are also used as means for specifically detecting and measuring trace substances, and are used in enzyme immunoassays, fluorescence immunoassays, and the like. These measurement methods have been applied to test drugs and have become indispensable for research in the fields of medicine, pharmacy, biochemistry, and the like.
[0004]
As a general method for preparing an antibody, a method in which an antiserum is prepared by inoculating an animal such as a mouse, a rabbit, or a sheep with an antigen and immunizing the same, and a polyclonal antibody is purified from the antiserum, Proliferating in the abdominal cavity, preparing ascites containing the monoclonal antibody, a method of purifying the antibody from the ascites fluid, culturing the hybridoma in a serum medium or serum-free medium to prepare a culture solution containing the monoclonal antibody, the culture solution And a method of purifying an antibody from the same.
[0005]
As a method for purifying the antibody from the antiserum or ascites fluid obtained as described above, the culture solution, purification methods such as salting out, ion exchange chromatography, gel filtration chromatography, and affinity chromatography are known. I have. For example, a gel in which protein A or protein G having a binding property to an IgG Fc fragment is immobilized as a ligand has been widely used for affinity chromatography.
[0006]
On the other hand, Non-Patent Document 1 reports peptides represented by SEQ ID NOs: 283 to 289 as peptides having a binding property to an Fc fragment of human IgG, and these peptides are used as antibodies for analysis or pharmaceutical use. It is described that it may be used as an affinity ligand in a purification step.
[0007]
[Non-patent document 1]
Journal of Immunological Methods 221 (1998) 151-157.
[0008]
[Problems to be solved by the invention]
However, depending on the type of animal producing the antibody, the above-mentioned protein A or protein G has no binding property to protein A or protein G even if IgG is used. (2) The binding between IgG and protein A (or protein G) is strong, and a strongly acidic eluent must be used to recover IgG, and denaturation or deactivation of IgG. (3) It is difficult to mass-produce protein A and protein G derived from fungi, the preparation cost is high, and (4) Contamination of contaminants derived from the cells of the raw material is a concern. However, there is a drawback in that it is difficult to use for the purification of antibodies used for pharmaceutical applications due to concerns about the safety of the antibody.
[0009]
On the other hand, since a peptide having a binding property to an IgG Fc fragment can be chemically synthesized, it can be mass-produced at low cost, and further, there is no need to worry about contaminants and the like. However, peptides other than the peptide described in Non-patent Document 1 described above have not been reported that have a binding property to an Fc fragment of IgG.
[0010]
Therefore, an object of the present invention is to provide a novel peptide or protein that has a binding property to an IgG Fc fragment and can be used for purification and detection of IgG.
[0011]
[Means for Solving the Problems]
In order to achieve the above object, the peptide or protein of the present invention has a modified amino acid sequence represented by SEQ ID NOS: 1 to 164, or a modified amino acid sequence having an amino acid residue within such an amino acid sequence that the binding to IgG Fc fragment is not impaired. It has an amino acid sequence selected from amino acid sequences in which a group has been substituted, inserted and / or deleted, and has a binding property to an IgG Fc fragment.
[0012]
According to the present invention, it is possible to provide a novel peptide or protein having a binding property to an IgG Fc fragment. These peptides or proteins include IgG purification, detection, and qualitative analysis (for example, confirmation of the loss of an antibody or selection of an IgG subclass, etc .; the same applies hereinafter), IgG Fc portion and Fc receptor. It can be used for research reagents, medicines, etc. by inhibiting the binding between them.
[0013]
In a preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOS: 1 to 87, or modifications, amino acid residue substitutions, insertions, and / or amino acid residues in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. Alternatively, there is provided a peptide or protein having an amino acid sequence selected from a deleted amino acid sequence and binding to an Fc fragment of human-derived IgG. These peptides or proteins can be used for purification, detection and qualification of human-derived IgG, research reagents, medicines, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0014]
In another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOs: 88 to 90, or those amino acid sequences are modified, substituted or inserted into amino acid residues within a range that does not impair the binding of IgG to the Fc fragment. And / or a peptide or protein having an amino acid sequence selected from a deleted amino acid sequence and having a binding property to a horse-derived IgG Fc fragment. These peptides or proteins can be used for purification, detection and qualification of horse-derived IgG, research reagents, medicines, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0015]
In still another preferred embodiment of the present invention, amino acid sequences represented by SEQ ID NOS: 91 to 93, or a modification thereof, amino acid residue substitution within the amino acid sequence thereof, as long as the binding to IgG Fc fragment is not impaired, A peptide or protein having an amino acid sequence selected from an inserted and / or deleted amino acid sequence and having a binding property to an Fc fragment of sheep-derived IgG is provided. These peptides or proteins can be used for purification, detection and qualification of sheep-derived IgG, research reagents, medicines and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0016]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOs: 94 to 97, or those amino acid sequences are modified or substituted with amino acid residues within a range that does not impair the binding to IgG Fc fragment. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted and / or deleted, and having a binding property to an Fc fragment of rabbit-derived IgG. These peptides or proteins can be used for purification, detection and qualification of rabbit-derived IgG, research reagents, medicaments, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0017]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOS: 98 to 104, or those amino acid sequences are modified and amino acid residues are substituted within a range that does not impair the binding to IgG Fc fragment. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted, deleted, and / or deleted, and having a binding property to an Fc fragment of guinea pig-derived IgG. These peptides or proteins can be used for purification, detection and qualification of guinea pig-derived IgG, as a research reagent, medicine and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0018]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOS: 105 to 108, or those amino acid sequences are modified or substituted with amino acid residues as long as the binding to IgG Fc fragment is not impaired. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted, deleted, and / or deleted, and having a binding property to an Fc fragment of goat-derived IgG. These peptides or proteins can be used for purification, detection and qualification of goat-derived IgG, research reagents, medicines, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0019]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOS: 109 to 131, or those amino acid sequences are modified or substituted with amino acid residues as long as the binding to IgG Fc fragment is not impaired. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted, deleted, and / or deleted, and having a binding property to an Fc fragment of cat-derived IgG. These peptides or proteins can be used for purification, detection and qualification of feline-derived IgG, research reagents, medicaments, and the like by inhibiting binding between the Fc portion of IgG and the Fc receptor.
[0020]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOs: 132 to 151, or the amino acid sequences thereof are modified or amino acid residues are modified within a range that does not impair the binding to IgG Fc fragment. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted, deleted, and / or deleted, and having a binding property to a dog-derived IgG Fc fragment. These peptides or proteins can be used for purification, detection and qualification of canine-derived IgG, research reagents, medicines, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0021]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOs: 152 to 156, or those amino acid sequences are modified or substituted with amino acid residues as long as the binding to IgG Fc fragment is not impaired. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted, deleted, and / or deleted and having a binding property to an Fc fragment of bovine IgG. These peptides or proteins can be used for purification, detection and qualification of bovine IgG, research reagents, medicines, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0022]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOs: 157 to 162, or those amino acid sequences are modified and amino acid residues are substituted within a range that does not impair the binding of IgG to the Fc fragment. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted and / or deleted and having a binding property to an Fc fragment of swine-derived IgG. These peptides or proteins can be used for purification, detection, and qualification of swine-derived IgG, as research reagents, drugs, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0023]
According to still another preferred embodiment of the present invention, the amino acid sequences represented by SEQ ID NOs: 163-164, or the amino acid sequences thereof are modified or substituted with amino acid residues within a range that does not impair the binding to IgG Fc fragment. And a peptide or protein having an amino acid sequence selected from amino acid sequences inserted, deleted and / or deleted, and having a binding property to a mouse-derived IgG Fc fragment. These peptides or proteins can be used for purification, detection and qualification of mouse-derived IgG, research reagents, medicines, and the like by inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
The peptide of the present invention is a peptide having an ability to bind to an Fc fragment of IgG, in which the peptide has an amino acid sequence represented by SEQ ID NOS: 1 to 164, or the amino acid sequence thereof has an impaired binding to the Fc fragment of IgG. It has an amino acid sequence selected from the amino acid sequences modified, substituted with amino acid residues, inserted and / or deleted to the extent that they are not present.
[0025]
The peptide of the present invention is not particularly limited in the number of amino acids as long as it has the above partial amino acid sequence, but is usually preferably composed of 4 to 50 amino acids, more preferably composed of 6 to 20 amino acids. Is more preferred. The peptide of the present invention may be modified (for example, acetylation, acylation, amidation, carboxylation, phosphorylation, addition of a sugar chain, etc.) or amino acid residue within a range that does not impair the binding of IgG to the Fc fragment. There may be substitutions, insertions and / or deletions. The peptide of the present invention may have a plurality of the above amino acid sequences.
[0026]
Further, the protein of the present invention is a protein having a binding property to an IgG Fc fragment, and the protein has an amino acid sequence represented by SEQ ID NOs: 1 to 164 or a binding property to an IgG Fc fragment in those amino acid sequences. Has an amino acid sequence selected from modified, amino acid residue substituted, inserted and / or deleted amino acid sequences as long as the amino acid sequence is not impaired, and the molecular weight and the like are not particularly limited. Must be displayed on the protein molecule so that it can contact and bind to the Fc fragment of IgG. The protein of the present invention may have a plurality of the above amino acid sequences.
[0027]
Examples of the protein of the present invention include the following.
{Circle around (1)} The peptide having a binding property to the IgG Fc fragment was bound to an enzyme such as HRP (horseradish peroxidase), AP (alkaline phosphatase), β-D-galactosidase, glucose-6-phosphate dehydrogenase, and the like. thing. According to a known method described in, for example, “Ultrasensitive Enzyme Immunoassay” (Eiji Ishikawa, Gakkai Shuppan Center), such a protein can be combined with the above-mentioned peptide having the binding property to the IgG Fc fragment and the above-mentioned enzyme. Can be obtained by bonding
[0028]
{Circle around (2)} A phage displaying on its surface a peptide having a binding property to the IgG Fc fragment.
Such phage are described in Smith, G .; P. , Science, 288, 1315-1317 (1985); K. Scott and G.S. P. Smith, Science, 249, 386-390 (1990) and the like. That is, a DNA encoding a peptide capable of binding to the IgG Fc fragment is chemically synthesized, and this DNA is inserted into a gene encoding a coat protein of the phage DNA in the same manner as in the case of preparing a phage library. Then, by introducing this DNA into E. coli, a phage displaying the desired peptide on the surface can be obtained.
[0029]
{Circle around (3)} An antibody in which the above-mentioned peptide having a binding property to the Fc fragment of IgG is linked to a predetermined antigen, and an antibody against the antigen is bound. Such an antibody can be obtained by the method described in Japanese Patent Application No. 2002-241688 filed by the present applicant. For example, a peptide capable of binding to the Fc fragment of IgG and a peptide serving as an antigen may be synthesized as one continuous peptide, and bound by an antigen-antibody reaction with an antibody against the antigen.
[0030]
The peptide of the present invention was obtained by a phage display method (Smith, GP, Science, 288, 1315-1317 (1985)) using an IgG Fc fragment as a target substance. (BioPackage). The phage display method binds to a target substance using a phage library in which a peptide having a random amino acid sequence (generally, about 5 to 12 amino acids) is displayed as a fusion protein on a phage coat protein. This is a method for screening peptides. Phage libraries are described, for example, in Smith, G .; P. , Science, 288, 1315-1317 (1985); K. Scott and G.S. P. According to the method described in Smith, Science, 249, 386-390 (1990), randomized DNA is chemically synthesized, and this is inserted into a gene encoding a coat protein of phage DNA. Can be prepared. In addition, a phage library is commercially available, and for example, a trade name “Phase Display Peptide Library Kit”, manufactured by New England Biolab) can be used.
[0031]
Then, based on the amino acid sequence determined by the phage display method or the like, the target peptide can be easily synthesized by a known peptide synthesis method such as the solid phase method or the Fmoc method.
[0032]
For example, the amino acid sequences represented by SEQ ID NOs: 1 to 87 are consensus sequences of peptides having binding properties to Fc fragments of human-derived IgG. These consensus sequences are obtained by contacting the phage library with a human-derived IgG Fc fragment to carry out a selection operation (biopanning) to obtain only a phage group that has expressed a peptide that binds to a human-derived IgG Fc fragment. This was determined by selecting and analyzing the DNA of the phage to identify the amino acid sequence of the peptide displayed on the phage surface and comparing the amino acid sequences of the obtained peptides (see Table 1 in Example 1). 2).
[0033]
Amino acid sequences represented by SEQ ID NOs: 1 to 87, or amino acid sequences selected from modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. Peptides or proteins having a modified amino acid sequence and binding to a human-derived IgG Fc fragment can be obtained by purifying, detecting, and qualifying human-derived IgG, a research reagent, a medicament for inhibiting binding between an Fc portion of an IgG and an Fc receptor. Etc. can be used. For example, by binding the above peptide to a suitable carrier, it can be used as a carrier for an affinity column for purifying human-derived IgG. In addition, by binding a labeling substance such as a fluorescent substance to the above peptide, it can be used as a reagent for detecting human-derived IgG.
[0034]
When the peptide is bound to a carrier or when a labeling substance is bound to the peptide, an appropriate spacer that can be used for binding to the carrier or the labeling substance may be inserted at the end of the peptide. Examples of the spacer include an amino acid, a peptide, a main chain having 2 to 18 carbon atoms (which may have an ester bond or an ether bond in the main chain), and preferably a hydrophilic functional group such as a hydroxyl group. Examples thereof include those having a group (for example, polyvinyl alcohol having an active group at both ends, preferably those obtained by polymerizing about 2 to 10 vinyl alcohol molecules), and sugar chains composed of 2 to 10 sugars. Preferred examples of the peptide include a flexible linker such as a peptide in which several (usually 2 to 10) glycines and serines are bonded to each other.
[0035]
By inserting such a spacer, binding to a carrier and binding to a labeling substance are facilitated, and by adjusting the length of the spacer, the peptide and the target substance (Fc fragment of human-derived IgG) can be combined with the peptide. Steric binding inhibition can be avoided, and the above-mentioned peptide can be more easily contacted with a target substance (Fc fragment of human-derived IgG), so that column performance and detection sensitivity can be improved. It becomes possible. For example, when the spacer is an amino acid or a peptide, the peptide with the inserted spacer can be synthesized as one continuous peptide, which is preferable.
[0036]
In addition, the amino acid sequences represented by SEQ ID NOs: 88 to 90 are common sequences of peptides having a binding property to the Fc fragment of horse-derived IgG. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by performing phage display in the same manner as described above, using a horse-derived IgG Fc fragment as a target substance (see See Table 4 in Example 6).
[0037]
Selected from amino acid sequences represented by SEQ ID NOs: 88 to 90 or modified, amino acid residue-substituted, inserted and / or deleted amino acid sequences in such an amino acid sequence as long as the binding to IgG Fc fragment is not impaired. The peptide or protein having the amino acid sequence of the present invention and having a binding property to the Fc fragment of horse-derived IgG can be bound to an appropriate carrier or a labeling substance, as described above, to obtain the horse-derived IgG. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting binding between the Fc portion of IgG and the Fc receptor.
[0038]
The amino acid sequences represented by SEQ ID NOs: 91 to 93 are common sequences of peptides having binding properties to the Fc fragment of sheep-derived IgG. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by performing phage display in the same manner as described above, using a sheep-derived IgG Fc fragment as a target substance (see (See Table 6 in Example 7).
[0039]
Selected from the amino acid sequences represented by SEQ ID NOs: 91 to 93, or amino acid sequences having modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or the protein having the modified amino acid sequence and binding to the Fc fragment of sheep-derived IgG can be bound to an appropriate carrier or a labeling substance, as described above, to obtain the sheep-derived IgG. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0040]
The amino acid sequences represented by SEQ ID NOs: 94 to 97 are peptides having a binding property to a rabbit IgG-derived Fc fragment. Phage display was performed in the same manner as described above using a rabbit-derived IgG Fc fragment as a target substance. The amino acid sequence was identified by the method (see Table 8 in Example 8).
[0041]
Selected from amino acid sequences represented by SEQ ID NOs: 94 to 97 or amino acid sequences in which the amino acid sequences have been modified, substituted with amino acid residues, inserted and / or deleted as long as the binding to IgG Fc fragments is not impaired. The peptide or protein having the amino acid sequence of the present invention and binding to the Fc fragment of rabbit IgG can be bound to an appropriate carrier or a labeling substance to bind the rabbit IgG as described above. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0042]
In addition, the amino acid sequences represented by SEQ ID NOs: 98 to 104 are common sequences of peptides having binding properties to guinea pig-derived IgG Fc fragments. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by performing phage display in the same manner as described above, using a guinea pig-derived IgG Fc fragment as the target substance. (See Table 10 in Example 9).
[0043]
Selected from the amino acid sequences represented by SEQ ID NOs: 98 to 104, or amino acid sequences in which the amino acid sequences are modified, substituted with amino acid residues, inserted and / or deleted as long as the binding of the amino acid sequence to the Fc fragment of IgG is not impaired. The peptide or protein having the amino acid sequence of the present invention and having a binding property to the Fc fragment of guinea pig-derived IgG can be bound to an appropriate carrier or a labeling substance, as described above, to obtain the guinea pig-derived IgG. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting binding between the Fc portion of IgG and the Fc receptor.
[0044]
The amino acid sequences represented by SEQ ID NOS: 105 to 108 are common sequences of peptides having a binding property to the Fc fragment of goat-derived IgG. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by performing phage display in the same manner as described above, using a goat-derived IgG Fc fragment as the target substance (see (See Table 12 in Example 10).
[0045]
Selected from amino acid sequences represented by SEQ ID NOS: 105 to 108, or amino acid sequences having modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. A peptide or protein having the amino acid sequence of the present invention and having a binding property to the Fc fragment of goat-derived IgG can be bound to an appropriate carrier or a labeling substance, as described above. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0046]
The amino acid sequences represented by SEQ ID NOS: 109 to 131 are common sequences of peptides having a binding property to the Fc fragment of cat-derived IgG. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by using the Fc fragment of cat-derived IgG as a target substance and performing phage display in the same manner as described above (see (See Table 14 in Example 11).
[0047]
Selected from amino acid sequences represented by SEQ ID NOS: 109 to 131 or amino acid sequences in which the amino acid sequences have been modified, substituted with amino acid residues, inserted and / or deleted as long as the binding to IgG Fc fragments is not impaired. The peptide or protein having the amino acid sequence of the present invention and having a binding property to the Fc fragment of cat-derived IgG can be bound to a suitable carrier or a labeling substance, as described above. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0048]
In addition, the amino acid sequences represented by SEQ ID NOs: 132 to 151 are common sequences of peptides having binding properties to Fc fragments of canine-derived IgG. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by performing phage display in the same manner as described above, using a dog-derived IgG Fc fragment as the target substance (see (See Table 16 in Example 12.)
[0049]
Amino acid sequences represented by SEQ ID NOs: 132 to 151, or amino acid sequences modified, substituted with amino acid residues, inserted and / or deleted in those amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein having the amino acid sequence shown above and binding to the Fc fragment of canine-derived IgG can be bound to a suitable carrier or a labeling substance, as described above, to obtain the canine-derived IgG. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0050]
The amino acid sequences represented by SEQ ID NOs: 152 to 156 are a common sequence of peptides having a binding property to an Fc fragment of bovine IgG. These consensus sequences were determined by using the Fc fragment of bovine IgG as a target substance and comparing the amino acid sequences of the peptides obtained by performing the phage display method in the same manner as described above (implementation). (See Table 18 in Example 13).
[0051]
Amino acid sequences represented by SEQ ID NOs: 152 to 156, or amino acid sequences modified, substituted with amino acid residues, inserted and / or deleted as long as the binding to IgG Fc fragments is not impaired in those amino acid sequences. The peptide or protein having the amino acid sequence of the present invention and having a binding property to the Fc fragment of bovine IgG can be bound to an appropriate carrier or a labeling substance in the same manner as described above. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0052]
In addition, the amino acid sequences represented by SEQ ID NOs: 157 to 162 are common sequences of peptides having binding properties to Fc fragments of swine-derived IgG. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by performing phage display in the same manner as described above, using a pig-derived IgG Fc fragment as the target substance (see (See Table 20 in Example 14).
[0053]
Amino acid sequences represented by SEQ ID NOs: 157 to 162, or amino acid sequences selected from modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein having the amino acid sequence of the present invention and binding to the Fc fragment of porcine-derived IgG can be bound to an appropriate carrier or a labeling substance in the same manner as described above. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting binding between the Fc portion of IgG and the Fc receptor.
[0054]
In addition, the amino acid sequences represented by SEQ ID NOs: 163-164 are common sequences of peptides having binding properties to mouse-derived IgG Fc fragments. These consensus sequences were determined by comparing the amino acid sequences of peptides obtained by performing phage display in the same manner as described above, using a mouse-derived IgG Fc fragment as the target substance (see (See Table 22 in Example 15).
[0055]
Selected from amino acid sequences represented by SEQ ID NOs: 163-164, or modified, amino acid residue-substituted, inserted and / or deleted amino acid sequences in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. A peptide or a protein having the amino acid sequence of the present invention and having a binding property to the Fc fragment of mouse-derived IgG can be bound to an appropriate carrier or a labeling substance in the same manner as described above. Can be used for research reagents, medicines, etc. by purifying, detecting, and qualitatively inhibiting the binding between the Fc portion of IgG and the Fc receptor.
[0056]
【Example】
Hereinafter, the present invention will be described specifically with reference to examples.
[0057]
Example 1
(1) Screening of a peptide having a binding property to a human-derived IgG Fc fragment (hereinafter abbreviated as human Fc)
Using human Fc as a target substance, screening of a peptide having human Fc-binding properties was performed by the phage display method as follows. In the phage display method, a library in which peptides are randomly displayed on the minor protein pIII on the surface of the M13 phage (a peptide library having 7, 10, and 12 random amino acids to be displayed) was obtained from Smith, G. et al. P. , Science, 288, 1315-1317 (1985); K. Scott and G.S. P. It was created based on the description of Smith, Science, 249, 386-390 (1990) and the like, and these three types of libraries were used.
[0058]
The target human Fc (ICN / CAPPEL, PURIFIED HUMAN IGG FC) is a magnetic bead coated with streptavidin, labeled with biotin using the trade name “sulfoNHS-LC-Biotin” (Pierce). (Dynabeads M280 streptavidin, manufactured by Dyanal) using biotin-streptavidin binding activity. The magnetic beads were blocked by suspending in a 2% (w / v) skim milk solution (10 mM phosphate buffer, pH 7.4) to minimize non-specific binding.
[0059]
Using these magnetic beads, a phage display method (selection operation three times) was performed according to a conventional method, and the amino acid sequence of a peptide having human Fc binding properties represented by SEQ ID NOS: 165 to 207 was identified.
[0060]
(2) Confirmation of the binding property of the obtained peptide to human Fc
Human Fc as a target was immobilized on a Corning ELISA plate (high binding type) as follows. human Fc (ICN / CAPPEL, PURIFIED HUMANIGG FC) was adjusted to a carbonic acid buffer (100 mM NaHCO3) at 100 μg / ml. ₃ , PH 8.0) was prepared, and 100 μl of this solution was added to each well of the plate and left at 4 ° C. overnight to immobilize the human Fc fragment. In the same manner as above, Fc fragments of various IgGs from chicken, rabbit, horse and goat (all manufactured by ROCKLAND) were immobilized on a Corning ELISA plate (high binding type) as a specificity control. did.
[0061]
The wells consisted of 24 columns, one column for anti-human Fc antibody (positive control), one column for phage presenting a peptide having no binding to human Fc (negative control), and the remaining 22 columns. Was used for a phage that exhibited a peptide having a binding property to each human Fc represented by SEQ ID NOs: 165 to 167, 169, 170, 172, 173, and 175 to 189.
[0062]
After leaving overnight, the solution in each well was discarded, and 300 μl of a blocking buffer (2% (w / v) skim milk PBS solution) was added to the Fc fragment-immobilized well and the empty well without target (for control, 24 μl). ) And left at room temperature for 2 hours.
[0063]
On the other hand, each phage (the above-mentioned 22 types of phage exhibiting a peptide having a binding property to human Fc and the phage presenting a peptide having no binding property to human Fc) solution (10 ¹¹ (pfu / μl) was added to 650 μl of the blocking buffer and left for 30 minutes to perform a pretreatment for reducing non-specific binding. In addition, 30 μl of anti-human Fc antibody (rabbitanti human IgG Fc antiserum, manufactured by Cappel) was dissolved in 650 μl of blocking buffer, and nonspecific adsorption reduction treatment was performed in the same manner.
[0064]
After discarding the blocking buffer in each well and washing 5 times with PBS-Tween (0.1% Tween in PBS), the above phage solution and anti-human Fc antibody solution were added to the human Fc-immobilized well and the animal species Fc 100 μl was added to each of the fragment-immobilized well and the control well, and the mixture was allowed to stand at room temperature for 1 hour with gentle stirring (about 20 rpm / min).
[0065]
Then, the solution in each well was discarded, and the well was washed six times with 200 μl of PBS-Tween. Then, 100 μl of a solution containing the following color-forming antibody was added to each well, and the mixture was allowed to stand for 1 hour with gentle stirring.
[0066]
-Well to which peptide-presenting phage was added: A solution in which a trade name "anti M13 antibody HRP monoclonalconjugate" (manufactured by Amersham Biosciences) was dissolved in a blocking buffer so as to be 4 µl / 20 ml.
[0067]
-Well to which anti-human Fc antibody was added: a solution obtained by dissolving trade name "HRP-anti rabbit antibody (Goat)" (manufactured by Cappel) in 2 ml of blocking buffer to a concentration of 0.2 µl / ml.
[0068]
Then, the solution in each well is discarded, and the well is washed six times with PBS-Tween. Then, an ABTS color developing solution (ABTS (manufactured by Wako Pure Chemical Industries, Ltd.) is adjusted to 0.22 mg / ml in a citrate buffer (50 mM, pH 4.0). ), And immediately before use, 200 μl of 30% hydrogen peroxide solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well at a concentration of 1.8 μl / ml to develop color. The absorbance (405 nm) was measured with a plate reader ("ARVO.SX", manufactured by Wallac). The result is shown in FIG. In FIG. 1, the Y-axis is obtained by dividing the absorbance of each Fc fragment-immobilized well by the absorbance of a control well.
[0069]
FIG. 1 shows that the peptides represented by SEQ ID NOs: 165 to 167, 169, 170, 172, 173, 175 to 189 have high specificity for human Fc. On the other hand, the antibody of the positive control binds to other than human Fc, indicating that polyclonal unabsorbed serum has low specificity.
[0070]
(3) For all of the obtained peptides (SEQ ID NOs: 165 to 207), the binding property to human Fc was examined by the same method (ELISA method) as in (2) above. Table 1 shows the results. The “coloring value” in Table 1 represents the absorbance of the human Fc (target substance) -immobilized well (405 nm) / the absorbance of the control (empty) well (405 nm). As the “coloring value” increases, the human Fc increases. Indicates that the affinity for is strong.
[0071]
[Table 1]

[0072]
From Table 1, it can be seen that the peptides represented by SEQ ID NOs: 165 to 207 have different affinities for human Fc, but all have binding ability to human Fc.
[0073]
(4) Search for the consensus sequence of the obtained peptide
From the amino acid sequences of the peptides represented by SEQ ID NOs: 165 to 207, a common sequence was searched for in a combination as shown in Table 2, and the common sequences represented by SEQ ID NOs: 1 to 87 were determined.
[0074]
[Table 2]

[0075]
Example 2
Example 1 was repeated except that human-derived IgG (manufactured by Cappel) was used in place of human Fc of Example 1 and various IgGs (manufactured by ROCKLAND) derived from pig, cow, mouse, goat, and rabbit were used as controls. An experiment was conducted in the same manner as in 1 (2), and the specificity of the peptides represented by SEQ ID NOs: 165 to 167, 169, 170, 172, 173, 175 to 189 for human-derived IgG was examined. The result is shown in FIG.
[0076]
As can be seen from FIG. 2, almost the same results as in the case of Example 1 were obtained, and the peptides represented by SEQ ID NOs: 165 to 167, 169, 170, 172, 173, 175 to 189 corresponded to human-derived IgG itself. It was also confirmed that the specificity was high.
[0077]
Example 3
Using the peptide represented by SEQ ID NO: 281 in which glycine and arginine were added to the C-terminal side of the peptide represented by SEQ ID NO: 166, it was confirmed whether human Fc could be purified. Glycine added to the C-terminal is used as a spacer, and arginine is used for adding an amino group for use in immobilization to a column using an amino group.
[0078]
The peptide represented by SEQ ID NO: 281 was requested to be synthesized by Sigma Genosis Co., Ltd. (80% purity guaranteed).
[0079]
<Immobilization to column>
In order to purify human Fc, a column on which the peptide represented by SEQ ID NO: 281 was immobilized was replaced with a trade name “HiTrap NHS-Activated HP (column volume 1 ml)” (manufactured by Amersham Biosciences, hereinafter simply referred to as a column). It was produced as follows.
[0080]
First, the column cap was removed, and the column was washed with 6 ml of ice-cold 1 mM HCl using a syringe, and then a solution containing the peptide represented by SEQ ID NO: 281 (1 mg / ml solution, buffer: 0.2 M carbonate buffer) NaHCO ₃ , 0.5 M NaCl, pH 8.3) was injected into the column with a syringe, and the mixture was allowed to stand at room temperature for 2 hours to immobilize the peptide on the column. After peptide immobilization, 6 ml of an ethanolamine solution (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and an acetate buffer (0.1 M acetic acid, 0 M) were used to block unreacted column functional groups and eliminate the activity. 6 ml of 0.5 M NaCl, pH 4.0) were passed alternately four times. In the meantime, after the second injection of the ethanolamine solution, the mixture was left at room temperature for 30 minutes. After this operation was completed, the column was equilibrated with 5 ml of PBS (10 mM phosphoric acid, 140 mM NaCl, pH 7.4).
[0081]
<Purification>
Using the column prepared as described above, human Fc was purified as follows. For purification, an HPLC system “AKTA purifier” (manufactured by Amersham Biosciences) was used.
[0082]
First, human Fc (PURIFIED HUMAN IGG FC manufactured by ICN / CAPPEL) was added to NaHCO so as to be 100 μg / ml. ₃ The solution was dissolved in a solution (100 mM, pH 8.0) to prepare a human Fc solution. As a control, a solution containing a pig-derived IgG Fc fragment (manufactured by ROCKLAND, SWINE IgG Fc fragment) which is said to be close to humans was prepared in the same manner. The column was then connected to an HPLC system and equilibrated with PBS at a flow rate of 1 ml / min (flow rate remained throughout the experiment). To this, 100 μl of the human Fc solution was injected and adsorbed on the column. After washing with 5 times the column volume (5 ml) of PBS, an eluent (PBS pH 3.0) was continuously supplied with a gradient. The gradient was set at 20 times the volume of the column (20 ml) until the eluent reached 100%. Further, a pig-derived IgG Fc fragment solution was applied to the column in the same manner as described above. FIG. 3 shows the elution results.
[0083]
Example 4
The peptide immobilized on the column was prepared in the same manner as in Example 3, except that the peptide represented by SEQ ID NO: 282 in which glycine and arginine were added to the C-terminal side of the peptide represented by SEQ ID NO: 173 was used. Was prepared, and human Fc was purified. The result is shown in FIG. It was confirmed by ELISA that the peptide represented by SEQ ID NO: 173 had lower affinity for human Fc than the peptide represented by SEQ ID NO: 166 used in Example 3 (Table 1). reference).
[0084]
Example 5
A column was prepared in the same manner as in Example 3 except that the peptide represented by SEQ ID NO: 281 and the peptide represented by SEQ ID NO: 282 were used as the peptide to be immobilized on the column, and purification of human Fc was performed. went. When these peptides were immobilized on the column, they were dissolved in a carbonate buffer to a concentration of 0.5 mg / ml (1 mg / ml in total) and immobilized. The result is shown in FIG.
[0085]
In FIG. 3, (A) shows the elution result of the column (control) not loaded with the sample, and (B) shows the elution result of the column immobilized with the peptide represented by SEQ ID NO: 281 when human Fc was loaded (implementation). Examples 3) and (C) show the elution results when a pig-derived IgG Fc fragment was loaded on a column on which the peptide represented by SEQ ID NO: 281 was immobilized (Example 3), and (D) shows the elution result represented by SEQ ID NO: 282 The elution results obtained when human Fc was loaded on the column on which the peptide was immobilized (Example 4), and (E) show that the human Fc was immobilized on the column on which the peptide represented by SEQ ID NO: 281 and the peptide represented by SEQ ID NO: 282 were immobilized. Indicates the elution results (Example 5) when (1) to (7) indicate the detected peaks.
[0086]
From the results of (B) and (C) in FIG. 3, it can be seen that human Fc is adsorbed on the column on which the peptide represented by SEQ ID NO: 281 is immobilized, but the pig-derived IgG Fc fragment is not adsorbed. . Therefore, it can be seen that human Fc can be specifically purified by using a peptide having a binding property to human Fc. In (B), multiple peaks ((1) to (3)) are considered to be due to differences in binding affinities of IgG subclasses IgG1 to IgG4, and a column for separating subclasses can also be prepared. Is suggested.
[0087]
On the other hand, from the results of (D) in FIG. 3, when a column to which the peptide represented by SEQ ID NO: 282 is immobilized is used, human Fc is eluted without using an eluent as in Example 3. You can see that. Therefore, it was shown that by using a peptide having low affinity for human Fc, IgG can be purified due to a difference in retention time, and it is possible to prevent inactivation or denaturation of IgG, which is a problem when purifying IgG. The possibility is suggested.
[0088]
In addition, the result of FIG. 3E shows that human Fc can be sufficiently purified even using a column to which the peptide represented by SEQ ID NO: 281 and the peptide represented by SEQ ID NO: 282 are immobilized. Since the state of the peaks (6) and (7) is different from the case where the above two peptides are used alone (in the case of Examples 3 and 4), the combination of the peptides is suitable for the purpose. It is also suggested that a suitable column can be prepared.
[0089]
Example 6
A phage display method (four selection operations) was performed in the same manner as in Example 1 (1) except that a horse-derived IgG Fc fragment (hereinafter, abbreviated to horse Fc) was used as a target substance. A peptide having a binding property to Fc was screened, and the amino acid sequences of the peptides represented by SEQ ID NOS: 208 to 212 were identified.
[0090]
For all the obtained peptides (SEQ ID NOS: 208 to 212), the binding to horse Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 3 shows the results. In Table 3, the "coloring value" represents the absorbance of the well Fc (target substance) immobilized well (405 nm) / the absorbance of the control (empty) well (405 nm). Indicates that the affinity is strong.
[0091]
[Table 3]

[0092]
From Table 3, it can be seen that the peptides represented by SEQ ID NOS: 208 to 212 have different affinities for horse Fc, but all have binding ability to horse Fc.
[0093]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 208 to 212, a common sequence was searched for in a combination as shown in Table 4, and the common sequences represented by SEQ ID NOs: 88 to 90 were determined.
[0094]
[Table 4]

[0095]
Example 7
A phage display method (selection operation 4 times) was carried out in the same manner as in Example 1 (1) except that a sheep-derived IgG Fc fragment (hereinafter abbreviated as sheep Fc) was used as a target substance. A peptide having a binding property to Fc was screened, and the amino acid sequences of the peptides represented by SEQ ID NOs: 213 to 216 were identified.
[0096]
For all the obtained peptides (SEQ ID NOs: 213 to 216), the binding property to the sheep Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 5 shows the results. The “coloring value” in Table 5 represents the absorbance (405 nm) of the well where the sheep Fc (target substance) is immobilized / the absorbance (405 nm) of the control (empty) well. The larger the “coloring value”, the greater the shape Fc Indicates that the affinity for is strong.
[0097]
[Table 5]

[0098]
From Table 5, it can be seen that the peptides represented by SEQ ID NOs: 213 to 216 have different affinities for sheep Fc, but all have binding power to sheep Fc.
[0099]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 213 to 216, a common sequence was searched for in a combination as shown in Table 6, and the common sequence represented by SEQ ID NOs: 91 to 93 was determined.
[0100]
[Table 6]

[0101]
Example 8
A phage display method (four selection operations) was performed in the same manner as in Example 1 (1) except that a rabbit-derived IgG Fc fragment (hereinafter abbreviated as rabbit Fc) was used as a target substance. A peptide having a binding property to Fc was screened, and the amino acid sequences of the peptides represented by SEQ ID NOs: 217 to 220 were identified.
[0102]
For all of the obtained peptides (SEQ ID NOs: 217 to 220), the binding property to rabbit Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 7 shows the results. The “coloring value” in Table 7 represents the absorbance (405 nm) of the well in which the rabbit Fc (target substance) is immobilized / the absorbance (405 nm) of the control (empty) well. The larger the “coloring value”, the more the rabbit color Fc Indicates that the affinity for is strong.
[0103]
[Table 7]

[0104]
Table 7 shows that the peptides represented by SEQ ID NOs: 217 to 220 have different affinities for rabbit Fc, but all have binding power to rabbit Fc.
[0105]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 217 to 220, a common sequence was searched for in a combination as shown in Table 8, and the common sequences represented by SEQ ID NOs: 94 to 97 were determined.
[0106]
[Table 8]

[0107]
Example 9
A phage display method (selection operation 4 times) was carried out in the same manner as in Example 1 (1) except that a guinea pig-derived IgG Fc fragment (hereinafter abbreviated as guinea pig Fc) was used as a target substance. A peptide having a binding property to guinea pig Fc was screened, and the amino acid sequence of the peptide represented by SEQ ID NOs: 221 to 227 was identified.
[0108]
For all the obtained peptides (SEQ ID NOs: 221 to 227), the binding property to guinea pig Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 9 shows the results. The “coloring value” in Table 9 represents the absorbance (405 nm) of the well in which the guinea pig Fc (target substance) is immobilized / the absorbance (405 nm) of the control (empty) well. Pig Fc has strong affinity.
[0109]
[Table 9]

[0110]
From Table 9, it can be seen that the peptides represented by SEQ ID NOs: 221 to 227 have different affinities for guinea pig Fc, but all have binding power to guinea pig Fc.
[0111]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 221 to 227, a common sequence was searched for in a combination as shown in Table 10, and the common sequence represented by SEQ ID NOs: 98 to 104 was determined.
[0112]
[Table 10]

[0113]
Example 10
The phage display method (selection operation 4 times) was performed in the same manner as in Example 1 (1) except that a goat-derived IgG Fc fragment (hereinafter abbreviated as goat Fc) was used as the target substance. A peptide having a binding property to Fc was screened to identify amino acid sequences represented by SEQ ID NOs: 228 to 232.
[0114]
For all the obtained peptides (SEQ ID NOs: 228 to 232), the binding property to goat Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 11 shows the results. The “coloring value” in Table 11 represents the absorbance of the goat Fc (target substance) -immobilized well (405 nm) / the absorbance of the control (empty) well (405 nm). Indicates that the affinity for is strong.
[0115]
[Table 11]

[0116]
From Table 11, it can be seen that the peptides represented by SEQ ID NOs: 228 to 232 have different affinities for goat Fc, but all have binding ability to goat Fc.
[0117]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 228 to 232, a common sequence was searched for in a combination as shown in Table 12, and the common sequence represented by SEQ ID NOS: 105 to 108 was determined.
[0118]
[Table 12]

[0119]
Example 11
A phage display method (four selection operations) was performed in the same manner as in (1) of Example 1, except that a cat-derived IgG Fc fragment (hereinafter abbreviated as cat Fc) was used as the target substance. A peptide having a binding property to Fc was screened, and the amino acid sequences of the peptides represented by SEQ ID NOs: 233 to 248 were identified.
[0120]
For all the obtained peptides (SEQ ID NOs: 233 to 248), the binding to cat Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 13 shows the results. The “coloring value” in Table 13 represents the absorbance of the cat Fc (target substance) -immobilized well (405 nm) / the absorbance of the control (empty) well (405 nm). As the “coloring value” becomes larger, the cat Fc becomes larger. Indicates that the affinity for is strong.
[0121]
[Table 13]

[0122]
From Table 13, it can be seen that the peptides represented by SEQ ID NOs: 233 to 248 have different affinities for cat Fc, but all have a binding ability to cat Fc.
[0123]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 233 to 248, a common sequence was searched for in a combination as shown in Table 14, and the common sequences represented by SEQ ID NOS: 109 to 131 were determined.
[0124]
[Table 14]

[0125]
Example 12
A phage display method (four selection operations) was performed in the same manner as in (1) of Example 1, except that a dog-derived IgG Fc fragment (hereinafter abbreviated as dog Fc) was used as a target substance. A peptide having a binding property to Fc was screened, and the amino acid sequence of the peptide represented by SEQ ID NOs: 249 to 261 was identified.
[0126]
For all the obtained peptides (SEQ ID NOs: 249 to 261), the binding property to dog Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 15 shows the results. The “coloring value” in Table 15 represents the absorbance (405 nm) of the well where the dog Fc (target substance) is immobilized / the absorbance (405 nm) of the control (empty) well. The larger the “coloring value”, the greater the dog Fc. Indicates that the affinity for is strong.
[0127]
[Table 15]

[0128]
From Table 15, it can be seen that the peptides represented by SEQ ID NOs: 249 to 261 have different affinities for dog Fc, but all have binding ability to dog Fc.
[0129]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 249 to 261, a common sequence was searched for in a combination as shown in Table 16, and the common sequences represented by SEQ ID NOs: 132 to 151 were determined.
[0130]
[Table 16]

[0131]
Example 13
A phage display method (selection operation four times) was performed in the same manner as in (1) of Example 1, except that a bovine IgG Fc fragment (hereinafter abbreviated as bovine Fc) was used as a target substance. A peptide having a binding property to Fc was screened, and the amino acid sequence of the peptide represented by SEQ ID NOs: 262 to 267 was identified.
[0132]
For all of the obtained peptides (SEQ ID NOs: 262 to 267), the binding to bovine Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 17 shows the results. The “coloring value” in Table 17 represents the absorbance (405 nm) of the bovine Fc (target substance) -immobilized well / the absorbance (405 nm) of the control (empty) well. The larger the “coloring value”, the greater the bovine Fc. Indicates that the affinity for is strong.
[0133]
[Table 17]

[0134]
From Table 17, it can be seen that the peptides represented by SEQ ID NOs: 262 to 267 have different affinities for bovine Fc, but all have binding power to bovine Fc.
[0135]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 262 to 267, a common sequence was searched for in a combination as shown in Table 18, and the common sequence represented by SEQ ID NOs: 152 to 156 was determined.
[0136]
[Table 18]

[0137]
Example 14
A phage display method (4 selection operations) was performed in the same manner as in (1) of Example 1, except that an Fc fragment of swine-derived IgG (hereinafter abbreviated as “spine Fc”) was used as the target substance. A peptide having a binding property to Fc was screened, and the amino acid sequence of the peptide represented by SEQ ID NOs: 268 to 277 was identified.
[0138]
For all the obtained peptides (SEQ ID NOs: 268 to 277), the binding to spine Fc was examined by the same method (ELISA method) as in Example 1 (2). Table 19 shows the results. The “coloring value” in Table 19 represents the absorbance (405 nm) of the well in which the spine Fc (target substance) is immobilized / the absorbance (405 nm) of the control (empty) well. The larger the “coloring value”, the greater the spine Fc Indicates that the affinity for is strong.
[0139]
[Table 19]

[0140]
From Table 19, it can be seen that the peptides represented by SEQ ID NOs: 268 to 277 have different affinities for spine Fc, but all have binding power to spine Fc.
[0141]
In addition, from the amino acid sequences of the peptides represented by SEQ ID NOs: 268 to 277, a common sequence was searched in combinations as shown in Table 20, and the common sequences represented by SEQ ID NOs: 157 to 162 were determined.
[0142]
[Table 20]

[0143]
Example 15
A phage display method (selection operation 4 times) was performed in the same manner as in Example 1, (1) except that a mouse-derived IgG Fc fragment (hereinafter abbreviated as mouse Fc) was used as a target substance. A peptide having a binding property to Fc was screened, and the amino acid sequence of the peptide represented by SEQ ID NOs: 278 to 280 was identified.
[0144]
For all the obtained peptides (SEQ ID NOs: 278 to 280), the binding property to mouse Fc was examined by the same method (ELISA method) as in Example 1 (2). The results are shown in Table 21. The “coloring value” in Table 21 represents the absorbance of the mouse Fc (target substance) -immobilized well (405 nm) / the absorbance of the control (empty) well (405 nm). Indicates that the affinity for is strong.
[0145]
[Table 21]

[0146]
Table 21 shows that the peptides represented by SEQ ID NOs: 278 to 280 have different affinities for mouse Fc, but all have binding power to mouse Fc.
[0147]
Further, from the amino acid sequences of the peptides represented by SEQ ID NOs: 278 to 280, a common sequence was searched for in a combination as shown in Table 22, and the common sequence represented by SEQ ID NOs: 163 to 164 was determined.
[0148]
[Table 22]

[0149]
"Sequence List Free Text"
SEQ ID NOs: 1 to 87: consensus sequences determined from peptides having a binding property to the Fc fragment of human-derived IgG obtained by the phage display method.
SEQ ID NOs: 88 to 90: common sequences determined from peptides having binding properties to Fc fragments of horse-derived IgG obtained by phage display method.
SEQ ID NOs: 91 to 93: common sequences determined from a peptide having a binding property to an Fc fragment of sheep-derived IgG obtained by a phage display method.
SEQ ID NOS: 94 to 97: amino acid sequences determined from a peptide having a binding property to an Fc fragment of rabbit IgG obtained by a phage display method.
SEQ ID NOs: 98 to 104: consensus sequences determined from peptides having binding properties to guinea pig-derived IgG Fc fragments obtained by phage display method.
SEQ ID NOS: 105 to 108: common sequence determined from a peptide having a binding property to a goat-derived IgG Fc fragment obtained by a phage display method.
SEQ ID NOS: 109 to 131: common sequences determined from peptides having a binding property to the Fc fragment of cat-derived IgG obtained by the phage display method.
SEQ ID NOS: 132 to 151: common sequence determined from a peptide having a binding property to a dog-derived IgG Fc fragment obtained by a phage display method.
SEQ ID NOS: 152 to 156: common sequence determined from a peptide having a binding property to a bovine IgG Fc fragment obtained by a phage display method.
SEQ ID NOS: 157 to 162: common sequence determined from a peptide having a binding property to an Fc fragment of porcine IgG obtained by phage display method.
SEQ ID NOs: 163 to 164: common sequence determined from a peptide having a binding property to an Fc fragment of mouse-derived IgG obtained by a phage display method.
SEQ ID NOs: 165 to 207: peptides obtained by phage display method and having binding properties to human-derived IgG Fc fragments.
SEQ ID NOS: 208 to 212: Peptides obtained by a phage display method and having a binding property to an Fc fragment of horse-derived IgG.
SEQ ID NOs: 213 to 216: peptides obtained by phage display and having a binding property to an Fc fragment of sheep-derived IgG.
SEQ ID NOs: 217 to 220: Peptides obtained by a phage display method and having a binding property to rabbit Fc fragments of IgG.
SEQ ID NOs: 221 to 227: Peptides obtained by a phage display method and having a binding property to an Fc fragment of guinea pig-derived IgG.
SEQ ID NOs: 228 to 232: Peptides having a binding property to goat-derived IgG Fc fragments obtained by a phage display method.
SEQ ID NOS: 233 to 248: peptides obtained by phage display method and having a binding property to Fc fragment of cat-derived IgG.
SEQ ID NOS: 249 to 261: Peptide obtained by phage display and having a binding property to a dog-derived IgG Fc fragment.
SEQ ID NOS: 262 to 267: Peptides having binding properties to bovine IgG Fc fragment obtained by phage display method.
SEQ ID NOs: 268-277: Peptide obtained by phage display method and having a binding property to Fc fragment of swine-derived IgG.
SEQ ID NOs: 278-280: Peptides having binding properties to mouse-derived IgG Fc fragments obtained by phage display method.
SEQ ID NO: 281: A peptide obtained by adding glycine and arginine to the C-terminal side of the peptide represented by SEQ ID NO: 166, which is a peptide used as an affinity ligand when purifying an Fc fragment of human-derived IgG.
SEQ ID NO: 282: Peptide having glycine and arginine added to the C-terminal side of the peptide represented by SEQ ID NO: 173, and used as an affinity ligand when purifying an Fc fragment of human-derived IgG.
SEQ ID NOs: 283 to 289: Peptides having a binding property to the Fc fragment of human-derived IgG described in Non-Patent Document 1.
[0150]
【The invention's effect】
As described above, according to the present invention, a novel peptide or protein having a binding property to an IgG Fc fragment of human, horse, sheep, rabbit, guinea pig, goat, cat, dog, cow, pig, mouse, etc. Can be provided. Since these peptides have high specificity for IgG Fc fragments derived from various animal species, purification, detection and qualification of IgG derived from each animal, research reagents, pharmaceuticals, etc. by inhibiting the binding between IgG Fc portion and Fc receptor Can be used for
[0151]
These peptides have the advantage that they can be chemically synthesized and can be prepared in large quantities at low cost. In addition, there is no risk of contamination or the like, so that there is an advantage that it can be easily used for purification of antibodies for medical use. For example, when the peptide provided by the present invention is used as an affinity ligand for IgG purification, a column capable of separating IgG subclasses may be produced. Further, by using a peptide having a low affinity for the Fc fragment of IgG, it is possible to prepare a column capable of purifying IgG with a difference in retention time, and it is possible to prevent inactivation or denaturation of IgG in the purification step. There is.
[0152]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing the results of a test for confirming the binding of the peptides represented by SEQ ID NOs: 165 to 167, 169, 170, 172, 173, 175 to 189 to human Fc.
FIG. 2 is a view showing the results of examining the specificity of the peptides represented by SEQ ID NOs: 165 to 167, 169, 170, 172, 173, 175 to 189 with respect to human-derived IgG.
FIG. 3 is a graph showing the elution results when human Fc was purified using a column on which the peptides represented by SEQ ID NOs: 281 and 282 were immobilized.

Claims (12)

Amino acid sequences represented by SEQ ID NOs: 1 to 164, or amino acid sequences selected from modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. A peptide or protein having a modified amino acid sequence and binding to an IgG Fc fragment. Amino acid sequences represented by SEQ ID NOs: 1 to 87, or amino acid sequences selected from modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of human-derived IgG. Selected from amino acid sequences represented by SEQ ID NOs: 88 to 90 or modified, amino acid residue-substituted, inserted and / or deleted amino acid sequences in such an amino acid sequence as long as the binding to IgG Fc fragment is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of horse-derived IgG. Selected from the amino acid sequences represented by SEQ ID NOs: 91 to 93, or amino acid sequences having modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of sheep-derived IgG. Selected from amino acid sequences represented by SEQ ID NOs: 94 to 97 or amino acid sequences in which the amino acid sequences have been modified, substituted with amino acid residues, inserted and / or deleted as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of IgG derived from rabbit. Selected from the amino acid sequences represented by SEQ ID NOs: 98 to 104, or amino acid sequences in which the amino acid sequences are modified, substituted with amino acid residues, inserted and / or deleted as long as the binding of the amino acid sequence to the Fc fragment of IgG is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of guinea pig-derived IgG. Selected from amino acid sequences represented by SEQ ID NOS: 105 to 108, or amino acid sequences having modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of goat-derived IgG. Selected from amino acid sequences represented by SEQ ID NOS: 109 to 131 or amino acid sequences in which the amino acid sequences have been modified, substituted with amino acid residues, inserted and / or deleted as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of cat-derived IgG. Amino acid sequences represented by SEQ ID NOs: 132 to 151, or amino acid sequences modified, substituted with amino acid residues, inserted and / or deleted in those amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of canine IgG. Amino acid sequences represented by SEQ ID NOs: 152 to 156, or amino acid sequences modified, substituted with amino acid residues, inserted and / or deleted as long as the binding to IgG Fc fragments is not impaired in those amino acid sequences. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of bovine IgG. Amino acid sequences represented by SEQ ID NOs: 157 to 162, or amino acid sequences selected from modifications, amino acid residue substitutions, insertions, and / or deletions in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to an Fc fragment of porcine IgG. Selected from amino acid sequences represented by SEQ ID NOs: 163-164, or modified, amino acid residue-substituted, inserted and / or deleted amino acid sequences in such amino acid sequences as long as the binding to IgG Fc fragments is not impaired. The peptide or protein according to claim 1, which has a modified amino acid sequence and has a binding property to a mouse-derived IgG Fc fragment.

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