JP2022160410A - 所定の標的に対して親和性を有するヒトリポカリン2(Lcn2、hNGAL)の突然変異タンパク質 - Google Patents
所定の標的に対して親和性を有するヒトリポカリン2(Lcn2、hNGAL)の突然変異タンパク質 Download PDFInfo
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Abstract
Description
Ther. 303, 540-548)、又は、例えば、連鎖球菌性プロテインGのもののような細菌性アルブミン結合ドメインのような、アルブミン結合タンパク質(Konig,T.and Skerra, A. (1998) J. Immunol. 方法s 218, 73-83)を含む。接合相手として用いられうるアルブミン結合ペプチドの別の例は、例えば、Cys-Xaa1-Xaa2-Xaa3-Xaa4-Cysの共通配列を有するものであり、米国特許出願第2003/0069395号又はDennisら(Dennisetal. (2002) J. Biol. Chem. 277, 35035-35043)に記載のように、Xaa1はAsp、Asn、Ser、Thr、又はTrpであり、Xaa2はAsn、Gln、His、Ile、Leu、又はLysであり、Xaa3はAla、Asp、Phe、Trp、又はTyrであり、Xaa4はAsp、Gly、Leu、Phe、Ser、又はThrである。
lipocalin fold.Biol.Chem.382,1335-1342参照。)の突然変異タンパク質、又は毒素である。
(a)ヒトリポカリン2をコードする核酸分子に、Lcn2の直鎖状ポリペプチド配列における配列位置96、100、及び106に対応する配列位置のうちのいずれかの少なくとも1つをコードするヌクレオチドトリプレットで、突然変異誘発を受けさせることによって、1つ又はそれ以上の突然変異タンパク質核酸分子を生じるステップを含む。
(b)(a)で得られた1つ以上の突然変異タンパク質核酸分子を適切な発現系で発現させるステップ、及び、
(c)所定の標的に対して検出可能な結合親和性を有する1つ又はそれ以上の突然変異タンパクを、選択及び/又は単離によって濃縮するステップ、を含んでもよい。
mixed oligonucleotides for random mutagenesis.Nucleic AcidsRes22,5600-5607)。
(i)所定の標的/リガンドとして、免疫学的ハプテンの特性が示される遊離型又はコンジュゲート型の化学化合物、ペプチド、タンパク質、又は、例えば多糖、核酸分子(例えば、DNA又はRNA)、若しくは全ウイルス粒子やウイロイド等の他の高分子からなる群より選択される化合物を提供するステップと、
(ii)上記標的/リガンドと、当該標的/リガンドに対し結合親和性を有する突然変異タンパク質との間で複合体の形成を可能にするように、複数の突然変異タンパク質を上記リガンドと接触させるステップと、
(iii)結合親和性を有さない、又は実質的に有さない突然変異タンパク質を除去するステップと、
を備える。
a)突然変異タンパク質を前記化合物と接合するステップと、
b)突然変異タンパク質/化合物の複合体を事前選択された部位に送達するステップと、
を備える。
また、他の実施形態において、本発明は、対象者において生体内撮像を行うために、本発明の突然変異タンパク質又は本発明の突然変異タンパク質が含まれる医薬組成物を、上記対象者に投与することを含む方法を特徴とする。その対象者は、上記のように定義されてもよい。
突然変異体Lcn2ファージディスプレイライブラリの構造
Lcn2変異のコンビナトリアルライブラリは、クローン化cDNA(Breustedtetal. (2006) Biochim. Biophys. Acta 1764, 161-173)に基づいて生成された。そのクローン化cDNAは、シングル不対チオール側鎖(Goetz et al. (2000) Biochemistry 39, 1935-1941)を除去するためにアミノ酸置換Cys87Serを運ぶとともに、第2のBstXl制限酵素認識部位を導入するためにアミノ酸置換Gln28Hisを運んだ。この領域の突然変異誘発及びポリメラーゼ連鎖反応(PCR)アセンブリは、基本的に(Besteetal. (1999) Proc. Natl. Acad. Sci. USA 96, 1898-1903、 Skerra (2001) J. Biotechnol.74,257-275)に公開された方法に従って行っており、このとき、図1に示すように、オリゴデオキシヌクレオチド(配列番号:1-10)とのワンポット増幅反応を使用した。オリゴデオキシヌクレオチドは、配列番号:1-4のプライマーがコード鎖に対応してかつそれぞれアミノ酸位置36、40、41、49、52、68、70、72、73、77、79、81、96、100、103、106、125、127、132、又は134で縮重したコドンを運ぶ一方、配列番号:5-8のプライマーが非コード鎖に対応して且つ縮重したコドンやアンチコドンを運ばないように設計された。配列番号:9及び配列番号:10の2つの隣接するプライマーは、過剰に使用され、組み立てられたランダム化遺伝子フラグメントの増幅に役立つ。上記のように(Schlehuberetal. (2000) J. Mol. Biol. 297, 1105-1120)、全てのPCRステップは、Go-TaqホットスタートDNAポリメラーゼ(Promega,Mannheim,Germany)を用いて行った。
Mol. Recognit. 14 (1), 79-86)に基づいて、OmpAシグナルペプチド、アンバーコドンに続く変性された成熟Lcn2、及び糸状バクテリオファージM13の遺伝子III外被タンパク質におけるC末端フラグメントからなる融合タンパク質、即ち、上記のようなビリン結合タンパク質(Besteetal., supra、 Skerra, supra)に類似する融合タンパク質をコードした。8.4μgの消化PCR産物と94μgの消化プラスミドDNAとのライゲーション混合物によってE.coliXLl-Blue(Bullocket al. (1987)Biotechniques 5,376-378)をエレクトロポレーションした後、1x1010の形質転換細胞を得た。
(実施例2)
異なるフォーマットでAβ標的の調製
成熟β-アミロイド配列(Dodel et al. (2003) Lancet Neurology 2, 215-220)のアミノ酸1~40に対応するAβ40ペプチド(配列番号:29)は、リジンスペーサ(Peptide Speciality Laboratory, Heidelberg, Germany)を介して接続されたC末端ビオチン基を有するとともに非標識の形(W.M.Keck Laboratory, New Haven, USA)で凍結乾燥されたペプチドの合成品として購入された。均一単量体のAβ40は5mg以下のペプチドを0.5mlの1,1,1,3,3,3-ヘキサフルオロ-2-プロパノール(HFIP、Sigma-Aldrich,Steinheim, Germany)に室温で少なくとも0.5時間溶解させることによって得られた。その後、HFIPをSpeedVac濃縮器で蒸発させ、Aβ40は冷水で15分間超音波処理(Bandelin,Sonorex,RK100, Germany)された後、適当量の蒸留水に溶解した。0.22μMフィルター(Spin-XCentrifugeTube filter、 Corning, USA)ろ過した後、タンパク質の濃度は、1490M-1cm-1の計算された吸光係数を用いて280nmで吸収測定によって検出された(Gasteigeretal. (2003) ExPASy: the proteomics server for in-depthproteinknowledge and analysis. Nucleic Acids Res. 31, 3784-3788, http://www.expasy.ch/tools/protparam.html)。
Steinheim, Germany)、Trx、及びMBPと同様に、モル比2:1(標識試薬: 標的タンパク質)でビオチン又はジゴキシゲニン(DIG)によって標識された。
(実施例3)
ファージディスプレイによるAβ40ペプチドに対する親和性を有するLcn2突然変異タンパク質の選択
各パニングサイクルのために、PBS(4mM KH2PO4、16mM Na2HPO4、115mMNaCl、pH7.4)における約1012組み換え型ファージミドは、PBS/T(0.1%(v/v)のTween20[ポリオキシエチレンソルビタンラウリン酸モノエステルを含むPBS、AppliChem, Darmstadt,Germany])において2%(w/v)BSAで1時間ブロッキングされた。50の部分及び25μLのストレプトアビジン被覆電磁ビーズ懸濁液(DynaビーズM-280Streptavidin; Dynal Biotech, Invitrogen, Karlsruhe,Germanyand Streptavidin Magnetic Particles; Roche Diagnostics,Mannheim,Germany)は、それぞれPBS/Tで洗浄され、さらにPBS/Tにおいて2%(w/v)BSAで1時間ブロックされた。25のブロックされたビーズは、ビーズの特異的なファージミドを除去するためにブロックされたファージミドの前吸着に用いられ、50のブロックされたビーズは、その後、選択サイクルに用いられる。
(実施例4)
ファージディスプレイによるTrx-Αβ28融合タンパク質に対して親和性を有するLcn2突然変異タンパク質の選択
各パニングサイクルのために、まず、PBS(4mMKH2PO4、16mMNa2HPO4、115mMNaCl、pH7.4)における約1012の組み換え型ファージミドをサイクル1及び2のPBS/T(0.1%(v/v)のTween20を含有するPBS)に2%(w/v)のBSAで1時間ブロックした。選択サイクル3以降、PBS/Tにおける2%(w/v)のスキムミルク(Sucofin, TSI, Zeven, Germany)をブロッキング試薬として用いた。50及び25μLのanti-DIG-IgG被覆電磁ビーズ懸濁液(EuropaBioproductsLtd, Cambridge, UK)は、PBS/Tで洗浄され、PBS/T又はスキムミルクに2%(w/v)のBSAでそれぞれ1時間ブロックした。
NaCl、pH7.5)に再懸濁し、LB-Cam plates(10g/L Bacto Tryptone、5g/LBactoYeast Extract、5g/L NaCl、15g/L Bacto Agar、35mg/L クロラムフェニコール、pH7.5)に播種し、そして、32℃で14~16時間培養した。次に、細胞をプレートから掻き取り、組換えファージミドの脱出や再増幅のために用いる。
(実施例5)
スクリーニングELISAによるAβに対して特異性を有するLcn2突然変異タンパク質の同定
実施例3に示すように、Aβ40とファージミド選択の4つのサイクル後、Lcn2突然変異タンパク質の濃縮したプールはphNGAL98(配列番号:27)にサブクローニングされ、E.coliスーパ株TG1-Fの形質転換に用いられ(a derivative of E.coliK12TGI (Kim et al. (2009) J. Am. Chem. Soc. 131, 3565-3576)、そして、スクリーニングELISAに供した。
SunnyVale, USA)に測定された。
(実施例6)
フィルターサンドイッチコロニースクリーニングアッセイによるTrx-Aβ28に対して特異性を有するLcn2突然変異タンパク質の同定
実施例4に示すように、Trx-Aβ28とファージミド選択の6つのサイクル後、突然変異したLcn2遺伝子カセットは、BstXI(Fermentas、St.Leon-Rot、Germany)によって、プラスミドphNGAL124(配列番号: 42)でサブクローニングされた。そのプラスミドphNGAL124(配列番号: 42)は、OmpAシグナルペプチドの融合物と、C末端Strep-tagII (Schmidt 及び Skerra (2007)Nat. Protoc. 2, 1528-1535)の後にアンバー終止コドンを有するLcn2コーディング領域と、連鎖球菌性のタンパク質G(Schlehuberら.(2000) J. Mol. Biol. 297, 1105-1120)からのアルブミン結合ドメイン(ABD)のための遺伝子とをコードする。
Aβ及びED-Bに特異的なLcn2突然変異タンパク質の可溶性物質の生成及びその精製
組み換え型Lcn2及びその突然変異タンパク質は、E.coli BL21(Studier及びMoffat(1986)J.Mol.Biol.189,113-130),E.coli W3110(Bachmann (1990) Microbiol.Rev.54,130-197),E.coli JM83 (Yanisch-Perron ら.(1985) Gene 33,103-119)又はE.coli supE strain TG1-F(アクリジニウムオレンジを用いてそのエピソームから取り除かれたE.coli K12 TGIの誘導体 [Kimら. (2009)J.Am.Chem.Soc.131,3565-3576])におけるペリプラズム分泌により産生された。可溶性タンパク質の発現のために、成熟Lcn2タンパク質を有するOmpAシグナルペプチド(配列番号:28)とC末端Strep-tag IIの融合体をコードするプラスミドphNGAL98(配列番号:27)が用いられた。そのプラスミドは、突然変異した遺伝子カセットの一方向性サブクローニングのための2つの互換性のないBstXI制限酵素認識部位を有する。
(実施例8)
ELISA試験における種々のAβ標的の結合活性の測定
C末端Strep-tag IIを有するLcn2突然変異タンパク質の選択的捕獲(Schmidt及びSkerra (2007) Nat.Protoc.2,1528-1535)のために、96-well MaxiSorp ポリスチレンマイクロタイタープレート(Nunc,Langenselbold,Germany)は、10μg/ml StrepMAB-Immo(IBA,Gottingen,Germany)のPBS溶液を用いて4℃で一晩中コートされ、3%(w/v)BSAのPBS/T溶液を用いて室温で1時間ブロッキングされた。PBS/Tを用いた3回の洗浄ステップの後、実施例7からの精製されたLcn2突然変異タンパク質の1μM溶液50μlが1時間全てのWellにアプライされた。洗浄後、実施例2からのビオチニル化標的Aβ40、ΜΒΡ-Aβ40又はTrx-Aβ28の希釈系列の50μlが加えられ、1時間インキュベートされた。ここで、実施例2からのOva、MBP及びTrxのビオチニル化型は、陰性対照タンパク質として用いられた。Wellは、再度洗浄され、結合した接合体は、50μlの1:5000でPBS/Tにより希釈されたExtrAvidin/AP接合体(Sigma-Aldrich,Steinheim,Germany)を用いて1時間検出され、続いて、100mM Tris/HCl,pH8.8,100mM NaCl,5mM MgCl2中の0.5mg/mlパラニトロフェニルリン酸塩(AppliChem,Darmstadt,Germany)100μlの存在下でシグナルが生成された。405nmでの吸収ΔΑ/Δtのタイムコースは、SpectraMax 250リーダー(Molecular Devices,SunnyVale,USA)で測定され、データは、KaleidaGraphソフトウェア(Synergy software,Reading,PA)を用いて以下の方程式に適合された。
ΔΑ=ΔAmaxx[L]tot/(KD+[L]tot)
上記式において、[L]totは、アプライされたリガンド接合体の濃度を表し、KDは解離定数である(Voss及びSkerra(1997)Protein Eng.10,975-982)。
この式では、更なるパラメータとして曲線の傾きp(Hill係数)を有する。
表面プラズモン共鳴(SPR)を介したAβへの結合活性の測定
Lcn2突然変異タンパク質のリアルタイム分析は、ランニングバッファーとしてPBS/T(0.005%(v/v)Tween20を含むPBS)を用いて、Biacore X system (Biacore,Uppsala,Sweden)で行われる。10mM Na酢酸塩,pH4.5に溶解した、実施例2のΜΒΡ-Aβ40の50μg/ml溶液は、標準のアミンカップリング化学を用いてCMD200Iチップ(Xantec,Dusseldorf,Germany)に固定されて、リガンド濃度は1455共鳴単位(RU)であった。実施例7の精製されたLcn2突然変異タンパク質のS1-A4及びUS7は、10μL/minの流速で、2nMから125nMまでの濃度範囲でアプライされた。センサーグラムは、エタノールアミンを用いて活性化及び抑制されたコントロールチャンネルで測定された対応する信号を差し引くことにより校正された。キネティクスデータの評価は、BIAevaluationソフトウェアV 3.0を用いてフィッティングすることにより行われた(Karlssonら.(1991) J.Immunol.Methods 145,229-240)。このステップの間、kon及びkoffの比率は、全体的にフィットされ、一方、最大反応差Rmaxは、各曲線に局所的にフィットされる。
(実施例10)
SPOT膜におけるエピトープマッピングを介したAβエピトープの決定
Lcn2突然変異タンパク質S1-A4及びUS7のAβエピトープのマッピングのためのSPOT膜(Amino-PEG500-UC540シート,100x150mm,
Mavis,Koln,Germany)は、MultiPep RS計測器(Intavis,Koln,Germany)及び同一の販売業者から得た活性化アミノ酸を用いて、Frank((2002) J.Immunol.Methods 26213-26)に記載されているように、自動化手順で準備された。Aβ40のアミノ酸配列(配列番号:29)は、一連のヘキサマー、デカマー及びペンタデカマーのそれぞれの1アミノ酸を転位して全体の配列をカバーして、膜において合成された。これらのペプチドのC末端は、膜に共有結合され、一方、それらのN末端は、アセチル化された。固定されたStrep-tag II は、検出方法の陽性対照として用いられた。
使用の前に、膜は、エタノールで1回、PBS(4mM KH2PO4,16mM Na2HPO4,115mM NaCl,pH 7.4)で3回洗浄し、3%(w/v)BSAのPBS/T溶液(0.1%(v/v)Tween20を含むPBS)で1時間ブロッキングされた。PBS/Tで3回洗浄した後、膜は、PBS/Tに溶解されたLcn2突然変異タンパク質S1-A4(50nm)、US7(100nm)又は野生型Lcn2(100nm)と共に1時間インキュベートされ、PBS/Tで3回洗浄された。その後、Strep-tag IIを介したタンパク質検出のために、膜は、PBS/Tが溶媒である1:1500希釈されたストレプトアビジン/AP接合体(GE Healthcare,Buckinghamshire,UK)と共に1時間インキュベートされた。その後、膜は、PBS/Tで3回洗浄され、APバッファー(100mM Tris/HCl,pH 8.8,100mM NaCl,5mM MgCl2)で1回洗浄された後、Schlehuberら.((2000)J.Mol.Biol.297,1105-1120)に記載されているように、5-ブロモ-4-クロロ-3-インドリルリン酸塩、4-トルイジン塩(BCIP、AppliChem,Darmstadt,Germany)及びニトロブルーテトラゾリウム(NBT、AppliChem,Darmstadt,Germany)のAPバッファー溶液を用いて発色反応を行った。
(実施例11)
ThT凝集アッセイにおけるAβへの親和性を有するLcn2突然変異タンパク質の機能分析
実施例2からの、可溶化され、均質な単量体の非ビオチニル化Aβ40に対して、様々なLcn2突然変異タンパク質の存在下又は非存在下において、チオフラビンT凝集アッセイを行った。チオフラビンT(ThT、Sigma-Aldrich,Steinheim,Germany)は、β-シートリッチのアミロイド原線維及びオリゴマー前駆体に特異的に結合するが、Aβペプチドに結合せず、それは、ThT蛍光の増大を伴う(Khurana ら.(2005)J.Struct.Biol.151,229-238)。
(実施例12)
エキストラドメインB(ED-B)を含む組み換え型フィブロネクチンフラグメントの調製
ヒトフィブロネクチンの3つの異なる組み換え型フラグメントは、選択のための標的として用いられた:単独のエキストラドメインB(ED-Bと呼ばれる、Zardiら.(1987)EMBO Journal,6,2337-2342)、その隣接するドメイン7及び8に関連して同一のドメイン(FN7B8と呼ばれる)、並びに従来型ドメイン7、8及び9を含み、ED-Bを欠失しているFN789(Carnemollaら. (1996), Int.J.Cancer,68,397-405、 Leahy ら. (1994) Proteins 19,48-54)。
virus genome,Cambridge University,EngLand)] 又はBL21(Studier及びMoffatt (1986), 189, 113-130)の細胞質における可溶性タンパク質として産生された。従って、発現プラスミドを有するE.coliの2-Lインキュベートは、100μg/mlアンピシリンを含むLB培地を用いて37℃において、中間対数期で増殖された。200μg/Lアンヒドロテトラサイクリン(Acros,Geel,Belgium)の添加後に、増殖が37℃で5時間から7時間続けられた。細胞は、遠心分離により濃縮され、35mlの氷冷クロマトグラフィーバッファー(40mM Hepes/NaOH, 1M NaCl,pH7.5)に再懸濁され、超音波処理(S250D,Branson,Danbury CT,USA)により溶解された。
Mannheim, Germany)を用いてジゴキシゲニン標識された。遊離ジゴキシゲニンを除去するために、タンパク質溶液は、40mM Tris/HCl,115
mM NaCl及び1mM EDTA, pH7.5を用いて平衡化されたPD-10脱塩カラム(GE Healthcare, Munich, Germany)にアプライされた。成功したジゴキシゲニン化及びタンパク質の完全性は、SDS-PAGE、ESI質量分析又は抗ジゴキシゲニン-AP,Fabフラグメント(Roche Diagnostics,Mannheim,Germany)を用いた染色を介したドットブロットにより確認された。
(実施例13)
ファージディスプレイによるED-Bに対する親和性を有するLcn2変異体の選択
全部で4つのファージミドディスプレイ選択サイクルは、実施例1に記載されたように、Sloning BioTechnology GmbH により最初に合成された合成遺伝子コレクションに基づくLcn2ランダムファージミドライブラリを用いて行われた。第1の選択サイクルのために、300μlのTBS/E(40mM Tris/HCl,115mM NaCl、及び1mM EDTAに溶解され、50mMベンズアミジンが添加された約5×1012の組み換え型ファージミドは、0.4%(v/v)Tween 20[ポリオキシエチレンソルビタンラウリン酸モノエステル、AppliChem,Darmstadt,Germany])を含むTBSに溶解された8%(w/v)BSA(Sigma-Aldrich,Munich,Germany)を100μl加えることにより30分間ブロッキングされた。その後、この溶液は、2%(w/v)BSAのTBS/T(0.1%(v/v)Tween20を含むTBS)溶液により1時間ブロッキングされた抗ジゴキシゲニンIgG電磁ビーズ(Europa Bioproducts,Cambridge,UK)と共に1時間インキュベートされ、400μlの0,1μΜジゴキシゲニン標識組み換え型FN7B8でチャージされた(実施例12を参照)。
グリシン/HCl,pH2.2を用いて8分間の第2溶出が行われた。溶出液は回収され、2M酢酸又は50μlの0.5M Tris塩基の適量をそれぞれ組み合わせて用い、すぐに中和され、対数増殖期のE.coli XLl-Blueに移すために用いられた。ファージミドは、以下の公開されたプロトコル(Besteら.(1999)PNAS 96, 1898-903、 Schlehuberら.(2000) J Mol
Biol 297 1109-20)の次のパニングステップの前に、力価が上げられ、増幅された。
(実施例14)
スクリーニングELISAによるED-Bに対する親和性を有するLcn2変異体の選択
実施例13に記載されたファージディスプレイ選択から生じた、標的タンパク質ED-Bに特異的に結合するLcn2突然変異タンパク質の濃縮は、スクリーニングELISAにより観察された。最後のパニングステップからのファスミドのプールされた調製液を用いて、突然変異遺伝子カセットが発現プラスミドphNGAL98のBstXIを介してサブクローニングされた。それは、E.coliにおけるペリプラズム生産のためのOmpAシグナルペプチドとC末端Strep-tagIIを有するLcn2コーディング領域との融合体をコードする(Schmidt及びSkerra (2007)Nat. Protoc.2,1528-1535)。96-wellプレートにおける個々のLcn2突然変異タンパク質の可溶性発現は、以下のように行われた:100μg/mlアンピシリンを含む100μlTB培地(Tartof及びHobbs(1987)Bethesda Research Laboratory Focus 9,12)に、ランダムに選択された単一コロニーが播かれ、37℃で5時間振とう培養した(500rpmから800rpm、 Thermomixer comfort、Eppendorf,Hamburg,Germany)。各クローンのために、100μlの新鮮培地に、この培養液の10μlを播き、37℃で1時間から2時間振とう培養された後、22℃まで温度を下げられた。2時間から4時間の更なる培養の後に、Lcn2突然変異タンパク質の発現が、0.2μg/mlアンヒドロテトラサイクリン(Acros,Geel,Belgium)を添加して12時間から14時間の対数増殖期細胞において誘導された。タンパク質のペリプラズム放出は、1mg/mlリゾチーム(Roche Diagnostics,Mannheim,Germany)を含む40μlの2×BBS(0.2M
ホウ酸塩/NaOH,160mM NaCl,1mM EDTA,pH8.0)を加えて室温で1時間振とう培養することにより生じた。溶解液は、40μlの10%w/v BSA(Applichem,Darmstadt,Germany)のTBS溶液及び0,5% Tween-20を用いて1時間ブロッキングされ、細胞破片は、10分間の遠心分離により粗抽出物から除去された。
Germany)の表面に吸着するために、50μlのTBSに溶解された100μg/mlタンパク質溶液が各wellに加えられ、4℃で一晩中インキュベートされた。E.coliからの粗抽出物への暴露の前に、TBS/Tを用いた3回の洗浄ステップの後に、wellはTBS/Tに溶解された2%(w/v)BSAを用いて室温で3時間ブロッキングされ、繰り返し洗浄された。ELISAのために、50μlの清澄な溶解液はwellにアプライされ、1時間インキュベートされ、続いて、TBS/Tを用いて3回洗浄された。結合したLcn2突然変異タンパク質は、4-ニトロフェニルリン酸塩基質 (pNPP, 0.5mg/ml、 AppliChem GmbH, Darmstadt, Germany)を用い、0.1M Tris/HCl,0.1M NaCl,5mM MgCl2,pH8.8に溶解されたストレプトアビジン-アルカリ-ホスファターゼ結合体(TBS/Tで1:1500希釈、 GE Healthcare,Munich,Germany)を用いて検出された(実施例16を参照)。
(実施例15)
Lcn2及びその変異体の可溶性タンパク質の産生及び精製
詳細は実施例7を参照。
(実施例16)
ELISAにおけるED-Bへの結合活性の測定
96-well Nunc Maxisorp プレート(Thermo Fisher Scientific,Langenselbold,Germany)の表面へのFN7B8又はFN789の吸着のために、TBS/Eに溶解された50μlの100μg/mlタンパク質溶液は、各wellに加えられ、室温で2時間インキュベートされた。さらに、コントロールタンパク質を含むために、ブランクwellは120μlのTBS/ETに溶解された2%(w/v)BSA(AppliChem,Darmstadt,Germany)に暴露された。3回の洗浄ステップの後に、TBS/ETに溶解された120μlの2%(w/v)BSA(AppliChem,Darmstadt,Germany)を用いて室温で2時間ブロッキングされ、繰り返し洗浄され、50μlの精製Lcn2突然変異タンパク質の希釈系列が加えられ、1時間インキュベートされた。wellは再度洗浄され、結合したLcn2突然変異タンパク質は、TBS/Tで1:1500希釈された50μlのストレプトアビジン-アルカリ-ホスファターゼ結合体(GE
Healthcare,Munich,Germany)を用いて1時間検出され、続いて、100mM Tris/HCl,100 mM NaCl,5mM MgCl2,pH 8.8が溶媒である50μlの0.5mg/mlパラニトロフェニルリン酸塩の存在下でシグナルを生成した。405nmにおける吸収ΔΑ/Δtの経時変化は、SpectraMax 250 リーダー(Molecular Devices,SunnyVale,CA)で測定され、データはKaleidaGraphソフトウェアを用いて下記の式に適応させた。
この式において、[L]totはアプライされたリガンド接合体の濃度を表し、KDは解離定数(Voss及びSkerra(1997)Protein Eng.10,975-982)を表す。Lcn2変異体N7A、N9B及びN10Dのそれぞれは、14.8nM(N7A)、40.1nM(N9B)、30.0nm(N7E)及び51.2(N10D)のKD値でFN7B8に特異的に結合するが、FN789又はBSAに結合しないことが分かった。
(実施例17)
表面プラズモン共鳴(SPR)を介した組み換え型フィブロネクチンFN7B8への結合活性の測定
Lcn2突然変異タンパク質のリアルタイム分析は、ランニングバッファーとして、HBS/ET(0.005%(v/v)Tween20を含む20mM Hepes,pH
7.5,150mM NaCl,1mM EDTA)を用いてBiacore X system(Biacore,Uppsala,Sweden)で実行された。10mM
Na酢酸塩,pH4.0が溶媒である200μg/ml組み換え型FN7B8溶液は、標準のアミンカップリング化学(Biacore,Uppsala,Sweden)を用いて、CMD200mセンサーチップ(XanTec bioanalytics,Duesseldorf,Germany)に固定されて、リガンド濃度は約500共鳴単位(RU)であった。精製Lcn2突然変異タンパク質は、2.5から最大で160nMの濃度において、25μl/minの流量でアプライされた。Theセンサーグラムは、コントロールチャンネルのために測定される対応するシグナルを差し引くことにより 校正された。それは、エタノールアミンを用いて活性化又は抑制された。キネティクスデータの評価は、BIAevaluationソフトウェアV4.1を用いた全体的なフィッティングにより行われた(Karlssonら.(1991) J.Immunol.Methods 145,229-240)。
(表2)表面プラズモン共鳴により測定された、選択したLcn2突然変異タンパク質のFN7B8に対するキネティクス結合データ
ED-BポジティブCaCo2細胞の免疫染色
ヒト結腸癌細胞(CaCo2細胞は、H.Daniel,Technische Universitat Miinchen,Germanyにより快く提供された;Pujuguet ら., Am.J.Pathol.148,579-592)は、細胞コンフルエンスが約50から70%となるまで37℃の湿気環境で、10%ウシ胎児血清、1x MEM非必須Lアミノ酸及び50μg/mlゲンタマイシン(PAA Laboratories,Pasching,Austria)が添加されたEarle塩及びL-グルタミンを含むMEMにおいて、Nunc Lab-TekTM II chamber
slideTM system(各スライドに4つのチャンバ、Thermo Fisher Scientific,Langenselbold,Germany)で培養された。カバースライドに付着された細胞単層は、PBS(Dulbecco’s Ca2+及びMg2+非含有、 PAA Laboratories, Pasching, Austria)を用いて洗浄され、続いて蒸留水で洗浄され、その後5μg/ml DAPI(4’,6-ジアミノ-2-フェニルインドール、Sigma-Aldrich,Munich,Germany)を含む氷冷メタノールを用いて5分間固定及び対比染色された。
(Cell Signaling Technology,Danvers,USA)を用いて検出された。
(実施例19)
H1-G1の配列位置36におけるフリーシステイン残基の置換によるAβ特異的Lcn2突然変異タンパク質H1GA及びH1GVの生成
Cys36は、部位特異的突然変異誘発によりAla又はValに置換された。そのために、PCRは縮重したオリゴデオキシヌクレオチドDH-4(配列番号:45)、第2のオリゴデオキシヌクレオチドJ08rev(配列番号:48)、及び鋳型としてLcn2突然変異タンパク質H1-G1をコードするプラスミドDNA(配列番号:37)を用いて行われた。増殖されたフラグメントは、発現プラスミドphNGAL98におけるBstXIを介してサブクローニングされ、個々の置換変異体を同定するために配列決定された。アミノ酸置換の導入により生じたLcn2変異体は、H1GA(配列番号:49,50)及びH1GV(配列番号:51,52)と名付けられた。
(実施例20)
高光学濃度を有するE.coli培養物を用いる新規のAβ特異的Lcn2突然変異タンパク質H1GA及びH1GVの可溶性物質及びその精製
組み換え型Lcn2及びその突然変異タンパク質は、E.coli JM83におけるペリプラズム分泌により産生された。可溶性タンパク質の発現のために、H1GA(配列番号:49)又はH1GV(配列番号:51)のいずれかをコードする対応するBstXI挿入を有するプラスミドphNGAL98が用いられた。
組み換え型タンパク質を含む上清が回収された。
(実施例21)
BiacoreX計測器での表面プラズモン共鳴を介したΜΒΡ-Aβ40に対する結合活性の測定
Lcn2突然変異タンパク質H1GA又はH1GVとΜΒΡ-Aβ40(配列番号:33)との相互作用のリアルタイム分析は、ランニングバッファーとしてPBS/T(0.005%(v/v)Tween20を含む4mM KH2PO4,16mM Na2HPO4, 115mM NaCl,pH7.4)を用いて、Biacore X systemで行われた。10mM Na酢酸塩,pH4.5を溶媒とした実施例2からの15μg/mlΜΒΡ-Aβ40溶液は、標準のアミンカップリング化学を用いてCMD200Iチップ(Xantec,Dusseldorf,Germany)に固定されて、リガンド濃度は1316共鳴単位(RU)であった。実施例20の精製Lcn2突然変異タンパク質H1GA及びH1GVは、20μL/minの流量で、4nMから128nMまでの濃度範囲でアプライされた。そのデータは、エタノールアミンを用いて活性化及び抑制されたコントロールチャンネルの測定された対応する信号を差し引くこと、及びバッファー注入の平均の測定信号を差し引くことにより2重参照された。キネティクスデータは、BIAevaluationソフトウェアV 3.0を用いて全体的に適合された(Karlssonら.(1991)J.Immunol.Methods 145,229-240)。
(実施例22)
Biacore T100計測器測定における表面プラズモン共鳴を介したAβ40の結合活性の測定
Lcn2突然変異タンパク質H1GAとAβ40(配列番号:29)との相互作用のリアルタイム分析は、ランニングバッファーとしてPBS/P(0.005%(v/v)Surfactant P20を含む4mM KH2PO4,16mM Na2HPO4,115mM NaCl,pH7.4)を用いて、Biacore T100 system (Biacore,Uppsala,Sweden)で行われた。10mM酢酸ナトリウム pH4.5を溶媒とした実施例2からの10μg/ml Aβ40溶液は、標準のアミンカップリング化学を用いて、CM5チップ(Biacore,Uppsala,Sweden)に固定化されて、リガンド濃度は325RUであった。実施例20の精製Lcn2突然変異タンパク質H1GAは、30μl/minの流量で、1nMから32nMまでの濃度範囲でアプライされた。H1GAの希釈系列は、konの情報を得るために、300sの結合及び解離時間の両方を用いて導入された。低koff比の正確な測定のために、最高濃度は、7200sの解離時間を用いて分析された。そのデータは、実施例21のように2重参照された。結合反応のkon及びkoffは、データ処理及びキネティクスフィッティングのためにBiacore T100 Evaluationソフトウェア V2.0.3を用いて全体のデータセットから決定された。そのデータは、1:1結合モデルを用いて、全体的に適合された。
(実施例23)
ThT凝集アッセイによるLcn2突然変異タンパク質H1GAの機能分析
チオフラビンT(ThT)凝集アッセイのために、合成Aβペプチド(配列番号:29)は、1,1,1,3,3,3-ヘキサフルオロ-2-プロパノール(HFIP、Sigma-Aldrich,Steinheim,Germany)に12時間溶解された。HFIPは、真空下で蒸発され、Aβは適量の蒸留水に溶解され、4℃で15分間超音波処理され、濾過された(Costar Spin-X遠心管フィルターセルロース酢酸塩膜,0.45μM;Corning Inc.,Corning,NY)。可溶化単量体Aβは、その後すぐに、凝集アッセイに用いられた。
(実施例24)
表面プラズモン共鳴(SPR)を介した組み換え型フィブロネクチン単一ドメインED-Bへの結合活性の測定測定
Lcn2突然変異タンパク質のリアルタイム相互作用分析は、ランニングバッファーとして、HBS/ET(0.005%(v/v)Tween 20を含む20mM Hepes,pH 7.5,150mM NaCl,1mM EDTA)を用いて、Biacore X計測器で行われた。10mM Na酢酸塩,pH40が溶媒である100μg/ml組み換え型ED-B溶液は、標準のアミンカップリング化学を用いて、CMD200mセンサーチップに固定化されて、リガンド濃度は約180共鳴単位(RU)であった。精製Lcn2突然変異タンパク質は、25μl/minの流量で、2.5から160nmまでの濃度でアプライされた。センサーグラムは、エタノールアミンを用いて活性化及び抑制されたコントロールチャンネルの測定された対応するシグナルを差し引くことにより校正された。キネティクスデータの評価は、BIAevaluationソフトウェア V4.1を用いて全体的にフィッティングすることにより行われた(Karlssonら.(1991)J.Immunol.Methods 145,229-240)。N10Dの場合において、異種検体競合反応モデルは、データフィッティングのために用いられ、2つの速度定数を生じた。
(表3)表面プラズモン共鳴により測定されたLcn2突然変異タンパク質のED-Bに対するキネティクス結合データ
SEQUENCE LISTING
<110> PIERIS PHARMACEUTICALS GMBH
<120> Muteins of human lipocalin 2 (Lcn2, hNGAL) with affinity for a
given target
<150> US 61/267,098
<151> 2009-12-07
<160> 52
<170> PatentIn version 3.3
<210> 1
<211> 88
<212> DNA
<213> Artificial
<220>
<223> NNK oligomer for positions 36, 40, 41, 49, 52
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22)..(22)
<223> k is g or t
<220>
<221> misc_feature
<222> (32)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (34)..(34)
<223> k is g or t
<220>
<221> misc_feature
<222> (35)..(36)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (37)..(37)
<223> k is g or t
<220>
<221> misc_feature
<222> (59)..(60)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (61)..(61)
<223> k is g or t
<220>
<221> misc_feature
<222> (68)..(69)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (70)..(70)
<223> k is g or t
<400> 1
gaagtggtat gtggtaggtn nkgcagggaa tnnknnkctc agagaagaca aagacccgnn 60
kaagatgnnk gccaccatct atgagctg 88
<210> 2
<211> 79
<212> DNA
<213> Artificial
<220>
<223> NNK oligomer for positions 68, 70, 72, 73, 77, 79, 81
<220>
<221> misc_feature
<222> (20)..(21)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (22)..(22)
<223> k is g or t
<220>
<221> misc_feature
<222> (26)..(27)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (28)..(28)
<223> k is g or t
<220>
<221> misc_feature
<222> (32)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (34)..(34)
<223> k is g or t
<220>
<221> misc_feature
<222> (35)..(36)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (37)..(37)
<223> k is g or t
<220>
<221> misc_feature
<222> (47)..(48)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (49)..(49)
<223> k is g or t
<220>
<221> misc_feature
<222> (53)..(54)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (55)..(55)
<223> k is g or t
<220>
<221> misc_feature
<222> (59)..(60)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (61)..(61)
<223> k is g or t
<400> 2
caagagctac aatgtcaccn nkgtcnnktt tnnknnkaag aagtgtnnkt acnnkatcnn 60
kacttttgtt ccaggttcc 79
<210> 3
<211> 68
<212> DNA
<213> Artificial
<220>
<223> NNK oligomer for positions 96, 100, 103, 106
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (21)..(21)
<223> k is g or t
<220>
<221> misc_feature
<222> (31)..(32)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (33)..(33)
<223> k is g or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (42)..(42)
<223> k is g or t
<220>
<221> misc_feature
<222> (49)..(50)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (51)..(51)
<223> k is g or t
<400> 3
ggcgagttca cgctgggcnn kattaagagt nnkcctggan nkacgagtnn kctcgtccga 60
gtggtgag 68
<210> 4
<211> 68
<212> DNA
<213> Artificial
<220>
<223> NNK oligomer for positions 125, 127, 132, 134
<220>
<221> misc_feature
<222> (19)..(20)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (21)..(21)
<223> k is g or t
<220>
<221> misc_feature
<222> (25)..(26)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (27)..(27)
<223> k is g or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (42)..(42)
<223> k is g or t
<220>
<221> misc_feature
<222> (46)..(47)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (48)..(48)
<223> k is g or t
<400> 4
gctatggtgt tcttcaagnn kgttnnkcaa aacagggagn nkttcnnkat caccctctac 60
gggagaac 68
<210> 5
<211> 45
<212> DNA
<213> Artificial
<220>
<223> PCR-Primer with fixed nucleotide sequences corresponding to the
non-coding strand
<400> 5
ggtgacattg tagctcttgt cttctttcag ctcatagatg gtggc 45
<210> 6
<211> 42
<212> DNA
<213> Artificial
<220>
<223> PCR-Primer with fixed nucleotide sequences corresponding to the
non-coding strand
<400> 6
gcccagcgtg aactcgcctg gctgggaacc tggaacaaaa gt 42
<210> 7
<211> 54
<212> DNA
<213> Artificial
<220>
<223> PCR-Primer with fixed nucleotide sequences corresponding to the
non-coding strand
<400> 7
cttgaagaac accatagcat gctggttgta gttggtgctc accactcgga cgag 54
<210> 8
<211> 64
<212> DNA
<213> Artificial
<220>
<223> PCR-Primer with fixed nucleotide sequences corresponding to the
non-coding strand
<400> 8
ggagaagcgg atgaagttct cctttagttc cgaagtcagc tccttggttc tcccgtagag 60
ggtg 64
<210> 9
<211> 40
<212> DNA
<213> Artificial
<220>
<223> 5' flanking PCR-Oligo biotinylated
<400> 9
ccaggacaac caattccatg ggaagtggta tgtggtaggt 40
<210> 10
<211> 40
<212> DNA
<213> Artificial
<220>
<223> 3' flanking PCR-Oligo biotinylated
<400> 10
ttcagggagg cccagagatt tggagaagcg gatgaagttc 40
<210> 11
<211> 4746
<212> DNA
<213> Artificial
<220>
<223> Phage display vector phNGAL102 with CamR used as backbone for
NNK-Library
<400> 11
ccataacgct cggttgccgc cgggcgtttt ttattggcca gatgattaat tcctaatttt 60
tgttgacact ctatcattgg tagagttatt ttaccactcc ctatcagtga tagagaaaag 120
tgaaatgaat agttcgacaa aaatctagat aacgagggca aaaaatgaaa aagacagcta 180
tcgcgattgc agtggctctg gctggcttcg ctaccgtagc gcaggcccag gactccacct 240
cagacctgat cccagcccca cctctgagca aggtccctct gcagcagaac ttccaggaca 300
accaattcca tgggaagtgg tatgtggtag gtctcgcagg gaatgcaatt ctcagagaag 360
acaaagaccc gcaaaagatg tatgccacca tctatgagct gaaagaagac aagagctaca 420
atgtcacctc cgtcctgttt aggaaaaaga agtgtgacta ctggatcagg acttttgttc 480
caggttccca gccaggcgag ttcacgctgg gcaacattaa gagttaccct ggattaacga 540
gttacctcgt ccgagtggtg agcaccaact acaaccagca tgctatggtg ttcttcaaga 600
aagtttctca aaacagggag tacttcaaga tcaccctcta cgggagaacc aaggagctga 660
cttcggaact aaaggagaac ttcatccgct tctccaaatc tctgggcctc cctgaaaacc 720
acatcgtctt ccctgtccca atcgaccagt gtatcgacgg cagcgctggt ggggcctaga 780
ctgttgaaag ttgtttagca aaaccccata cagaaaattc atttactaac gtctggaaag 840
acgacaaaac tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg 900
ttgtagtttg tactggtgac gaaactcagt gttacggtac atgggttcct attgggcttg 960
ctatccctga aaatgagggt ggtggctctg agggtggcgg ttctgagggt ggcggttctg 1020
agggtggcgg tactaaacct cctgagtacg gtgatacacc tattccgggc tatacttata 1080
tcaaccctct cgacggcact tatccgcctg gtactgagca aaaccccgct aatcctaatc 1140
cttctcttga ggagtctcag cctcttaata ctttcatgtt tcagaataat aggttccgaa 1200
ataggcaggg ggcattaact gtttatacgg gcactgttac tcaaggcact gaccccgtta 1260
aaacttatta ccagtacact cctgtatcat caaaagccat gtatgacgct tactggaacg 1320
gtaaattcag agactgcgct ttccattctg gctttaatga ggatccattc gtttgtgaat 1380
atcaaggcca atcgtctgac ctgcctcaac ctcctgtcaa tgctggcggc ggctctggtg 1440
gtggttctgg tggcggctct gagggtggtg gctctgtggg tggcggttct gagggtggcg 1500
gctctgaggg aggcggttcc ggtggtggct ctggttccgg tgattttgat tatgaaaaga 1560
tggcaaacgc taataagggg gctatgaccg aaaatgccga tgaaaacgcg ctacagtctg 1620
acgctaaagg caaacttgat tctgtcgcta ctgattacgg tgctgctatc gatggtttca 1680
ttggtgacgt ttccggcctt gctaatggta atggtgctac tggtgatttt gctggctcta 1740
attcccaaat ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc 1800
aatatttacc ttccctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta 1860
aaccatatga attttctatt gattgtgaca aaataaactt attccgtggt gtctttgcgt 1920
ttcttttata tgttgccacc tttatgtatg tattttctac gtttgctaac atactgcgta 1980
ataaggagtc ttaataagct tgacctgtga agtgaaaaat ggcgcacatt gtgcgacatt 2040
ttttttgtct gccgtttacc gctactgcgt cacggatctc cacgcgccct gtagcggcgc 2100
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 2160
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 2220
tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 2280
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 2340
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 2400
aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 2460
ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 2520
ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat 2580
aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa 2640
aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga 2700
acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga 2760
tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat 2820
tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg 2880
tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga 2940
aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata 3000
ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga 3060
gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt 3120
ggccaatatg gacaacttct tcgcccccgt tttcactatg ggcaaatatt atacgcaagg 3180
cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca 3240
tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta 3300
ataggaatta atgatgtctc gtttagataa aagtaaagtg attaacagcg cattagagct 3360
gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt 3420
agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc 3480
cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca 3540
agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg 3600
agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca 3660
attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc 3720
agtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga 3780
agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt 3840
atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg 3900
attagaaaaa caacttaaat gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg 3960
atggtaactt cactagttta aaaggatcta ggtgaagatc ctttttgata atctcatgac 4020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 4080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 4140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 4200
aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg 4260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 4320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 4380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 4440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 4500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 4560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 4620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 4680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgac 4740
ccgaca 4746
<210> 12
<211> 4963
<212> DNA
<213> Artificial
<220>
<223> Phage display vector phNGAL108 with AmpR used for Sloning Library
<400> 12
ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt 60
tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct 120
agataacgag ggcaaaaaat gaaaaagaca gctatcgcga ttgcagtggc tctggctggc 180
ttcgctaccg tagcgcaggc ccaggactcc acctcagacc tgatcccagc cccacctctg 240
agcaaggtcc ctctgcagca gaacttccag gacaaccaat tccatgggaa gtggtatgtg 300
gtaggtctcg cagggaatgc aattctcaga gaagacaaag acccgcaaaa gatgtatgcc 360
accatctatg agctgaaaga agacaagagc tacaatgtca cctccgtcct gtttaggaaa 420
aagaagtgtg actactggat caggactttt gttccaggtt cccagccagg cgagttcacg 480
ctgggcaaca ttaagagtta ccctggatta acgagttacc tcgtccgagt ggtgagcacc 540
aactacaacc agcatgctat ggtgttcttc aagaaagttt ctcaaaacag ggagtacttc 600
aagatcaccc tctacgggag aaccaaggag ctgacttcgg aactaaagga gaacttcatc 660
cgcttctcca aatctctggg cctccctgaa aaccacatcg tcttccctgt cccaatcgac 720
cagtgtatcg acggcagcgc ttggcgtcac ccgcagttcg gtggggccta gactgttgaa 780
agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa 840
actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtagtt 900
tgtactggtg acgaaactca gtgttacggt acatgggttc ctattgggct tgctatccct 960
gaaaatgagg gtggtggctc tgagggtggc ggttctgagg gtggcggttc tgagggtggc 1020
ggtactaaac ctcctgagta cggtgataca cctattccgg gctatactta tatcaaccct 1080
ctcgacggca cttatccgcc tggtactgag caaaaccccg ctaatcctaa tccttctctt 1140
gaggagtctc agcctcttaa tactttcatg tttcagaata ataggttccg aaataggcag 1200
ggggcattaa ctgtttatac gggcactgtt actcaaggca ctgaccccgt taaaacttat 1260
taccagtaca ctcctgtatc atcaaaagcc atgtatgacg cttactggaa cggtaaattc 1320
agagactgcg ctttccattc tggctttaat gaggatccat tcgtttgtga atatcaaggc 1380
caatcgtctg acctgcctca acctcctgtc aatgctggcg gcggctctgg tggtggttct 1440
ggtggcggct ctgagggtgg tggctctgag ggtggcggtt ctgagggtgg cggctctgag 1500
ggaggcggtt ccggtggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac 1560
gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa 1620
ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac 1680
gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa 1740
atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta 1800
ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat 1860
gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta 1920
tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag 1980
tcttaataag cttgacctgt gaagtgaaaa atggcgcaca ttgtgcgaca ttttttttgt 2040
ctgccgttta ccgctactgc gtcacggatc tccacgcgcc ctgtagcggc gcattaagcg 2100
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2160
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 2220
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 2280
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 2340
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 2400
tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 2460
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 2520
ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 2580
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 2640
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 2700
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 2760
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 2820
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 2880
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 2940
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 3000
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 3060
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 3120
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 3180
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 3240
gcgaactact tactctagct tcccggcaac aattgataga ctggatggag gcggataaag 3300
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 3360
gagccggtga gcgtggctct cgcggtatca ttgcagcact ggggccagat ggtaagccct 3420
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 3480
agatcgctga gataggtgcc tcactgatta agcattggta ggaattaatg atgtctcgtt 3540
tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag 3600
gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc 3660
atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc 3720
atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta 3780
aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac 3840
ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag 3900
gtttttcact agagaatgca ttatatgcac tcagcgcagt ggggcatttt actttaggtt 3960
gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg 4020
atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc 4080
cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg 4140
aaagtgggtc ttaaaagcag cataaccttt ttccgtgatg gtaacttcac tagtttaaaa 4200
ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 4260
cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 4320
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 4380
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 4440
taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 4500
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 4560
agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 4620
gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 4680
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 4740
ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 4800
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 4860
tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 4920
ggttcctggc cttttgctgg ccttttgctc acatgacccg aca 4963
<210> 13
<211> 907
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> XbaI - HindIII cassette; Start-ATG: Position 43; FN7B8:
recombinant ED-B domain flanked by adjacent type III FN homology
repeats encoded as a cassette cloned on pASK75
<400> 13
tctagaaata attttgttta actttaagaa ggagatatac atatgccatt gtctccacca 60
acaaacttgc atctggaggc aaaccctgac actggagtgc tcacagtctc ctgggagagg 120
agcaccaccc cagacattac tggttataga attaccacaa cccctacaaa cggccagcag 180
ggaaattctt tggaagaagt ggtccatgct gatcagagct cctgcacttt tgataacctg 240
agtcccggcc tggagtacaa tgtcagtgtt tacactgtca aggatgacaa ggaaagtgtc 300
cctatctctg ataccatcat cccagaagtt ccgcagctga cagatctgtc cttcgttgac 360
atcaccgaca gctccatcgg tctgcgttgg accccgctga attcctccac catcatcggt 420
tatcgtatca ccgttgttgc tgctggtgaa gggatcccga tctttgaaga cttcgttgac 480
tcctccgttg gttactacac cgttaccggt ctggaacccg ggatcgacta cgacatctcc 540
gttatcaccc tgatcaacgg tggtgaatcc gctccgacca ccttaaccca gcagaccgcg 600
gttcctcctc ccactgacct gcgattcacc aacattggtc cagacaccat gcgtgtcacc 660
tgggctccac ccccatccat tgatttaacc aacttcctgg tgcgttactc acctgtgaaa 720
aatgaggaag atgttgcaga gttgtcaatt tctccttcag acaatgcagt ggtcttaaca 780
aatctcctgc ctggtacaga atatgtagtg agtgtctcca gtgtctacga acaacatgag 840
agcacacctc ttagaggaag acagaaaaca ggtagcgctc accatcacca tcaccattaa 900
taagctt 907
<210> 14
<211> 298
<212> PRT
<213> Homo sapiens
<220>
<221> MISC_FEATURE
<223> XbaI - HindIII cassette; Start-MET: Position 13; FN7B8:
recombinant ED-B domain flanked by adjacent type III FN homology
repeats encoded as a cassette cloned on pASK75
<400> 14
Ser Arg Asn Asn Phe Val Leu Glu Gly Asp Ile His Met Pro Leu Ser
1 5 10 15
Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr Gly Val Leu
20 25 30
Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile Thr Gly Tyr Arg
35 40 45
Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly Asn Ser Leu Glu Glu
50 55 60
Val Val His Ala Asp Gln Ser Ser Cys Thr Phe Asp Asn Leu Ser Pro
65 70 75 80
Gly Leu Glu Tyr Asn Val Ser Val Tyr Thr Val Lys Asp Asp Lys Glu
85 90 95
Ser Val Pro Ile Ser Asp Thr Ile Ile Pro Glu Val Pro Gln Leu Thr
100 105 110
Asp Leu Ser Phe Val Asp Ile Thr Asp Ser Ser Ile Gly Leu Arg Trp
115 120 125
Thr Pro Leu Asn Ser Ser Thr Ile Ile Gly Tyr Arg Ile Thr Val Val
130 135 140
Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu Asp Phe Val Asp Ser Ser
145 150 155 160
Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu Pro Gly Ile Asp Tyr Asp
165 170 175
Ile Ser Val Ile Thr Leu Ile Asn Gly Gly Glu Ser Ala Pro Thr Thr
180 185 190
Leu Thr Gln Gln Thr Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr
195 200 205
Asn Ile Gly Pro Asp Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser
210 215 220
Ile Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu
225 230 235 240
Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val
245 250 255
Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser
260 265 270
Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr
275 280 285
Gly Ser Ala His His His His His His Ala
290 295
<210> 15
<211> 379
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> XbaI - HindIII cassette; Start-ATG: Position 43; ED-B:
Recombinant ED-B domain alone encoded as a cassette cloned on
pASK75
<400> 15
tctagaaata attttgttta actttaagaa ggagatatac aaatgcgtgg ttccgaagtt 60
ccgcagctga cagatctgtc cttcgttgac atcaccgaca gctccatcgg tctgcgttgg 120
accccgctga attcctccac catcatcggt tatcgtatca ccgttgttgc tgctggtgaa 180
gggatcccga tctttgaaga cttcgttgac tcctccgttg gttactacac cgttaccggt 240
ctggaacccg ggatcgacta cgacatctcc gttatcaccc tgatcaacgg tggtgaatcc 300
gctccgacca ccttaaccca gcagaccgct gggagcgctc accatcacca tcaccattaa 360
gggagccacc cgcaagctt 379
<210> 16
<211> 123
<212> PRT
<213> Homo sapiens
<220>
<221> MISC_FEATURE
<223> XbaI - HindIII cassette; Start-MET: Position 13; ED-B:
Recombinant ED-B domain alone encoded as a cassette cloned on
pASK75
<400> 16
Ser Arg Asn Asn Phe Val Leu Glu Gly Asp Ile Gln Met Arg Gly Ser
1 5 10 15
Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr Asp Ser
20 25 30
Ser Ile Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser Thr Ile Ile Gly
35 40 45
Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu
50 55 60
Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu
65 70 75 80
Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly Gly
85 90 95
Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr Ala Gly Ser Ala His
100 105 110
His His His His His Gly Ser His Pro Gln Ala
115 120
<210> 17
<211> 904
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> XbaI - HindIII cassette; Start-ATG: Position 43; FN789:
recombinant Type III FN homology repeats without ED-B encoded as
a cassette cloned on pASK75
<400> 17
tctagaaata attttgttta actttaagaa ggagatatac atatgccatt gtctccacca 60
acaaacttgc atctggaggc aaaccctgac actggagtgc tcacagtctc ctgggagagg 120
agcaccaccc cagacattac tggttataga attaccacaa cccctacaaa cggccagcag 180
ggaaattctt tggaagaagt ggtccatgct gatcagagct cctgcacttt tgataacctg 240
agtcccggcc tggagtacaa tgtcagtgtt tacactgtca aggatgacaa ggaaagtgtc 300
cctatctctg ataccatcat cccagctgtt cctcctccca ctgacctgcg attcaccaac 360
attggtccag acaccatgcg tgtcacctgg gctccacccc catccattga tttaaccaac 420
ttcctggtgc gttactcacc tgtgaaaaat gaggaagatg ttgcagagtt gtcaatttct 480
ccttcagaca atgcagtggt cttaacaaat ctcctgcctg gtacagaata tgtagtgagt 540
gtctccagtg tctacgaaca acatgagagc acacctctta gaggaagaca gaaaacaggt 600
cttgattccc caactggcat tgacttttct gatattactg ccaactcttt tactgtgcac 660
tggattgctc ctcgagccac catcactggc tacaggatcc gccatcatcc cgagcacttc 720
agtgggagac ctcgagaaga tcgggtgccc cactctcgga attccatcac cctcaccaac 780
ctcactccag gcacagagta tgtggtcagc atcgttgctc ttaatggcag agaggaaagt 840
cccttattga ttggccaaca atcaacagtt agcgctcacc atcaccatca ccattaataa 900
gctt 904
<210> 18
<211> 297
<212> PRT
<213> Homo sapiens
<220>
<221> MISC_FEATURE
<223> XbaI - HindIII cassette; Start-MET: Position 13; FN789:
recombinant Type III FN homology repeats without ED-B encoded as
a cassette cloned on pASK75
<400> 18
Ser Arg Asn Asn Phe Val Leu Glu Gly Asp Ile His Met Pro Leu Ser
1 5 10 15
Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp Thr Gly Val Leu
20 25 30
Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile Thr Gly Tyr Arg
35 40 45
Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly Asn Ser Leu Glu Glu
50 55 60
Val Val His Ala Asp Gln Ser Ser Cys Thr Phe Asp Asn Leu Ser Pro
65 70 75 80
Gly Leu Glu Tyr Asn Val Ser Val Tyr Thr Val Lys Asp Asp Lys Glu
85 90 95
Ser Val Pro Ile Ser Asp Thr Ile Ile Pro Ala Val Pro Pro Pro Thr
100 105 110
Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val Thr Trp
115 120 125
Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg Tyr Ser
130 135 140
Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser Pro Ser
145 150 155 160
Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu Tyr Val
165 170 175
Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu Arg
180 185 190
Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe Ser
195 200 205
Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg Ala
210 215 220
Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe Ser Gly
225 230 235 240
Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile Thr Leu
245 250 255
Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile Val Ala Leu
260 265 270
Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser Thr Val
275 280 285
Ser Ala His His His His His His Ala
290 295
<210> 19
<211> 534
<212> DNA
<213> Artificial
<220>
<223> N7A: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 19
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcaaagc cggaaatcac 120
gacctgcgtg aggataagga tccgcgtaaa atgcaagcga ccatttacga gttgaaagaa 180
gataaatcat ataacgtcac caatgtgcgt tttgttcaca agaaatgcaa ttaccgtatt 240
tggacctttg tgccggggag ccagccgggc gagtttactt taggcaatat taaaagttgg 300
ccgggcctga catcatggtt ggtccgcgtc gtgagcacca actacaacca gcatgccatg 360
gtgttcttca agcgtgtgta ccagaaccgc gagctgtttg agatcacact gtacgggcgc 420
acgaaagaac tgacaaacga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 20
<211> 178
<212> PRT
<213> Artificial
<220>
<223> N7A: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 20
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Lys Ala Gly Asn His Asp Leu Arg Glu Asp Lys Asp Pro
35 40 45
Arg Lys Met Gln Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Asn Val Arg Phe Val His Lys Lys Cys Asn Tyr Arg Ile
65 70 75 80
Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn
85 90 95
Ile Lys Ser Trp Pro Gly Leu Thr Ser Trp Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Arg Val Tyr Gln
115 120 125
Asn Arg Glu Leu Phe Glu Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Asn Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 21
<211> 534
<212> DNA
<213> Artificial
<220>
<223> N7E: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 21
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcgaggc cggaaatagc 120
ctgctgcgtg aggataagga tccgcgtaaa atgtacgcga ccatttacga gttgaaagaa 180
gataaatcat ataacgtcac cagcgtgcgt tttcgtagca agaaatgcca ctacctgatt 240
cgtacctttg tgccggggag ccagccgggc gagtttactt taggcctgat taaaagtaaa 300
ccgggccaca catcattctt ggtccgcgtt gtgagcacca actacaacca gcatgccatg 360
gtgttcttca agaccgtggc acagaaccgc gagtactttt tcatcacact gtacgggcgc 420
acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 22
<211> 178
<212> PRT
<213> Artificial
<220>
<223> N7E: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 22
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Glu Ala Gly Asn Ser Leu Leu Arg Glu Asp Lys Asp Pro
35 40 45
Arg Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Arg Phe Arg Ser Lys Lys Cys His Tyr Leu Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Leu
85 90 95
Ile Lys Ser Lys Pro Gly His Thr Ser Phe Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Thr Val Ala Gln
115 120 125
Asn Arg Glu Tyr Phe Phe Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 23
<211> 534
<212> DNA
<213> Artificial
<220>
<223> N9B: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 23
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggccgtgc cggaaatatg 120
cgtctgcgtg aggataagga tccggcaaaa atggttgcga ccatttacga gttgaaagaa 180
gataaatcat ataacgtcac caaagtgatg tttcaacgta agaaatgcaa atacatgatt 240
aatacctttg tgccggggag ccagccgggc gagtttactt taggcgcaat taaaagtcct 300
ccgggcccta catcaacctt ggtccgcgtc gtgagcacca actacaacca gcatgccatg 360
gtgttcttca agcacgtgtt ccagaaccgc gagtactttc acatcacact gtacgggcgc 420
acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 24
<211> 178
<212> PRT
<213> Artificial
<220>
<223> N9B: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 24
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Arg Ala Gly Asn Met Arg Leu Arg Glu Asp Lys Asp Pro
35 40 45
Ala Lys Met Val Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Lys Val Met Phe Gln Arg Lys Lys Cys Lys Tyr Met Ile
65 70 75 80
Asn Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Ala
85 90 95
Ile Lys Ser Pro Pro Gly Pro Thr Ser Thr Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys His Val Phe Gln
115 120 125
Asn Arg Glu Tyr Phe His Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 25
<211> 534
<212> DNA
<213> Artificial
<220>
<223> N10D: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 25
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcgcagc cggaaatacc 120
tggctgcgtg aggataagga tccgtacaaa atgcaagcga ccatttacga gttgaaagaa 180
gataaatcat ataacgtcac caatgtgctg tttatgagca agaaatgccg ttacatgatt 240
cacacctttg tgccggggag ccagccgggc gagtttactt taggcagcat taaaagttgg 300
ccgggcctga catcatggtt ggtccgcgtc gtgagcacca actacaacca gcatgccatg 360
gtgttcttca agcgtgtgta ccagaaccgc gagttctttg gaatcacact gtacgggcgc 420
acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 26
<211> 178
<212> PRT
<213> Artificial
<220>
<223> N10D: Mutein of hNGAL specific for ED-B of fibronectin
cloned on phNGAL98
<400> 26
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Ala Ala Gly Asn Thr Trp Leu Arg Glu Asp Lys Asp Pro
35 40 45
Tyr Lys Met Gln Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Asn Val Leu Phe Met Ser Lys Lys Cys Arg Tyr Met Ile
65 70 75 80
His Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Ser
85 90 95
Ile Lys Ser Trp Pro Gly Leu Thr Ser Trp Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Arg Val Tyr Gln
115 120 125
Asn Arg Glu Phe Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 27
<211> 3745
<212> DNA
<213> Artificial
<220>
<223> Expression vector phNGAL98 with AmpR encoding wild type Lcn2 with
the C-terminal Strep-tagII
<400> 27
ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt 60
tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct 120
agataacgag ggcaaaaaat gaaaaagaca gctatcgcga ttgcagtggc tctggctggc 180
ttcgctaccg tagcgcaggc ccaggactcc acctcagacc tgatcccagc cccacctctg 240
agcaaggtcc ctctgcagca gaacttccag gacaaccaat tccatgggaa gtggtatgtg 300
gtaggtctcg cagggaatgc aattctcaga gaagacaaag acccgcaaaa gatgtatgcc 360
accatctatg agctgaaaga agacaagagc tacaatgtca cctccgtcct gtttaggaaa 420
aagaagtgtg actactggat caggactttt gttccaggtt cccagccagg cgagttcacg 480
ctgggcaaca ttaagagtta ccctggatta acgagttacc tcgtccgagt ggtgagcacc 540
aactacaacc agcatgctat ggtgttcttc aagaaagttt ctcaaaacag ggagtacttc 600
aagatcaccc tctacgggag aaccaaggag ctgacttcgg aactaaagga gaacttcatc 660
cgcttctcca aatctctggg cctccctgaa aaccacatcg tcttccctgt cccaatcgac 720
cagtgtatcg acggcagcgc ttggtctcac ccgcagttcg aaaaataata agcttgacct 780
gtgaagtgaa aaatggcgca cattgtgcga catttttttt gtctgccgtt taccgctact 840
gcgtcacgga tctccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 900
cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 960
cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 1020
tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 1080
gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 1140
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 1200
attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 1260
tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca 1320
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1380
tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 1440
gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 1500
ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 1560
cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 1620
ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 1680
cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 1740
tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 1800
tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 1860
tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 1920
ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 1980
tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 2040
cttcccggca acaattgata gactggatgg aggcggataa agttgcagga ccacttctgc 2100
gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggct 2160
ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 2220
acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 2280
cctcactgat taagcattgg taggaattaa tgatgtctcg tttagataaa agtaaagtga 2340
ttaacagcgc attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac 2400
tcgcccagaa gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg 2460
ctttgctcga cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt 2520
tagaagggga aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt 2580
tactaagtca tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt 2640
atgaaactct cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg 2700
cattatatgc actcagcgca gtggggcatt ttactttagg ttgcgtattg gaagatcaag 2760
agcatcaagt cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat 2820
tacgacaagc tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc 2880
ttgaattgat catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcttaaaagc 2940
agcataacct ttttccgtga tggtaacttc actagtttaa aaggatctag gtgaagatcc 3000
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 3060
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 3120
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 3180
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 3240
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 3300
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 3360
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 3420
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 3480
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 3540
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3600
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3660
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 3720
ggccttttgc tcacatgacc cgaca 3745
<210> 28
<211> 178
<212> PRT
<213> Artificial
<220>
<223> Wild type Lcn2 cloned on phNGAL98 without OmpA signal peptide and
Strep-tagII
<400> 28
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro
35 40 45
Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn
85 90 95
Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln
115 120 125
Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 29
<211> 40
<212> PRT
<213> Homo sapiens
<220>
<221> MISC_FEATURE
<223> peptide sequence of A beta 40
<400> 29
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys
1 5 10 15
Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile
20 25 30
Gly Leu Met Val Gly Gly Val Val
35 40
<210> 30
<211> 11
<212> PRT
<213> Homo sapiens
<220>
<221> MISC_FEATURE
<223> peptide sequence of A beta 1-11
<400> 30
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu
1 5 10
<210> 31
<211> 12
<212> PRT
<213> Homo sapiens
<220>
<221> MISC_FEATURE
<223> peptide sequence of A beta 16-27
<400> 31
Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn
1 5 10
<210> 32
<211> 1182
<212> DNA
<213> Artificial
<220>
<223> XbaI HindIII DNA restriction fragment of pMBP-His. Reading frame
starts at position 27 with atg
<220>
<221> misc_feature
<222> (34)..(34)
<223> c can be t
<220>
<221> misc_feature
<222> (35)..(35)
<223> t can be c
<400> 32
tctagaacca acaaggacca tagcatatga aaactgaaga aggtaaactg gtaatctgga 60
ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata 120
ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg 180
cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg 240
ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc 300
cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg 360
aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag 420
agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc 480
tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt 540
atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg 600
gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact 660
ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg 720
catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca 780
agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc 840
cgaacaaaga gctggcgaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg 900
aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt 960
tggcgaaaga tccacgtatt gccgccacca tggaaaacgc ccagaaaggt gaaatcatgc 1020
cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg 1080
ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactcgt atcacccagg 1140
gatccctcga gatcaaacat caccaccatc accattaagc tt 1182
<210> 33
<211> 1308
<212> DNA
<213> Artificial
<220>
<223> XbaI HindIII DNA restriction fragment pASK75-MBP-Abeta40. Reading
frame starts at position 27 with atg
<220>
<221> misc_feature
<222> (34)..(34)
<223> c can be t
<220>
<221> misc_feature
<222> (35)..(35)
<223> t can be c
<400> 33
tctagaacca acaaggacca tagcatatga aaactgaaga aggtaaactg gtaatctgga 60
ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata 120
ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg 180
cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg 240
ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc 300
cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg 360
aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag 420
agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc 480
tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt 540
atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg 600
gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact 660
ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg 720
catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca 780
agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc 840
cgaacaaaga gctggcgaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg 900
aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt 960
tggcgaaaga tccacgtatt gccgccacca tggaaaacgc ccagaaaggt gaaatcatgc 1020
cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg 1080
ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactcgt atcacccagg 1140
gatcccatca ccaccatcat cacgagaact tgtacttcca ggacgctgaa ttccgtcacg 1200
actccggtta cgaagttcac caccagaagc tggttttctt cgctgaagac gttggttcca 1260
acaaaggtgc tatcatcggt ctgatggttg gtggtgttgt ttaagctt 1308
<210> 34
<211> 488
<212> DNA
<213> Artificial
<220>
<223> XbaI HindIII DNA restriction fragment of pASK75-TrxAbeta28H6.
Reading frame starts at position 23 with atg
<400> 34
tctagatact gtggagttat atatgagcga taaaattatt cacctgactg acgacagttt 60
tgacacggat gtactcaaag cggacggggc gatcctcgtc gatttctggg cagagtggtg 120
cggtccgggt ggtggtgacg ctgaattccg tcacgactcc ggttacgaag ttcaccacca 180
gaaactggtg ttcttcgctg aagacgttgg ttccaacaaa ggtggcggtc cgtgcaaaat 240
gatcgccccg attctggatg aaatcgctga cgaatatcag ggcaaactga ccgttgcaaa 300
actgaacatc gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg gtatcccgac 360
tctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac tgtctaaagg 420
tcagttgaaa gagttcctcg acgctaacct ggccagcgct caccatcacc atcaccatta 480
ataagctt 488
<210> 35
<211> 383
<212> DNA
<213> Artificial
<220>
<223> XbaI HindIII DNA restriction fragment of pASK75-TrxH6. Reading
frame starts at position 23 with atg
<400> 35
tctagatact gtggagttat atatgagcga taaaattatt cacctgactg acgacagttt 60
tgacacggat gtactcaaag cggacggggc gatcctcgtc gatttctggg cagagtggtg 120
cggtccgtgc aaaatgatcg ccccgattct ggatgaaatc gctgacgaat atcagggcaa 180
actgaccgtt gcaaaactga acatcgatca aaaccctggc actgcgccga aatatggcat 240
ccgtggtatc ccgactctgc tgctgttcaa aaacggtgaa gtggcggcaa ccaaagtggg 300
tgcactgtct aaaggtcagt tgaaagagtt cctcgacgct aacctggcca gcgctcacca 360
tcaccatcac cattaataag ctt 383
<210> 36
<211> 534
<212> DNA
<213> Artificial
<220>
<223> DNA sequence for the mature protein wt Lcn2 without Strep-tag II.
Reading frame starts with the first nucleotide of the following
sequence
<400> 36
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaag tggtatgtgg taggtctcgc agggaatgca 120
attctcagag aagacaaaga cccgcaaaag atgtatgcca ccatctatga gctgaaagaa 180
gacaagagct acaatgtcac ctccgtcctg tttaggaaaa agaagtgtga ctactggatc 240
aggacttttg ttccaggttc ccagccaggc gagttcacgc tgggcaacat taagagttac 300
cctggattaa cgagttacct cgtccgagtg gtgagcacca actacaacca gcatgctatg 360
gtgttcttca agaaagtttc tcaaaacagg gagtacttca agatcaccct ctacgggaga 420
accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 37
<211> 534
<212> DNA
<213> Artificial
<220>
<223> DNA sequence for the mature protein H1-G1 without Strep-tag II.
Reading frame starts with the first nucleotide of the following
sequence
<400> 37
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaag tggtatgtgg taggttgtgc agggaatgtg 120
ttgctcagag aagacaaaga cccgcttaag atgtatgcca ccatctatga gctgaaagaa 180
gacaagagct acaatgtcac cagtgtcggg tttgatgata agaagtgttt gtacaagatc 240
cggacttttg ttccaggttc ccagccaggc gagttcacgc tgggcaggat taagagtgag 300
cctggaggta cgagttggct cgtccgagtg gtgagcacca actacaacca gcatgctatg 360
gtgttcttca aggaggttgc gcaaaacagg gagacgttca atatcaccct ctacgggaga 420
accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 38
<211> 534
<212> DNA
<213> Artificial
<220>
<223> DNA sequence for the mature protein S1-A4 without Strep-tag II.
Reading frame starts with the first nucleotide of the following
sequence
<400> 38
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaag tggtatgtgg taggtgttgc agggaattat 120
acgctcagag aagacaaaga cccgctgaag atgtatgcca ccatctatga gctgaaagaa 180
gacaagagct acaatgtcac cagtgtcggg tttaggttga agaagtgtaa ttacaagatc 240
cggacttttg ttccaggttc ccagccaggc gagttcacgc tgggcattat taagagtcag 300
cctggaatga cgagttatct cgtccgagtg gtgagcacca actacaacca gcatgctatg 360
gtgttcttca agacggttgg gcaaaacagg gagatgttca atatcaccct ctacgggaga 420
accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 39
<211> 178
<212> PRT
<213> Artificial
<220>
<223> S1-A4 (Abeta)
<400> 39
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Val Ala Gly Asn Tyr Thr Leu Arg Glu Asp Lys Asp Pro
35 40 45
Leu Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Gly Phe Arg Leu Lys Lys Cys Asn Tyr Lys Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Ile
85 90 95
Ile Lys Ser Gln Pro Gly Met Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Thr Val Gly Gln
115 120 125
Asn Arg Glu Met Phe Asn Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 40
<211> 534
<212> DNA
<213> Artificial
<220>
<223> DNA sequence for the mature protein US7 without Strep-tag II.
Reading frame starts with the first nucleotide of the following
sequence
<400> 40
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaag tggtatgtgg taggtgttgc agggaataag 120
tctctcagag aagacaaaga cccgtggaag atgtatgcca ccatctatga gctgaaagaa 180
gacaagagct acaatgtcac ctcggtcggg tttgggacta agaagtgtca ttacaagatc 240
aggacttttg ttccaggttc ccagccaggc gagttcacgc tgggcaggat taagagtcgg 300
cctggaagga cgagtgctct cgtccgagtg gtgagcacca actacaacca gcatgctatg 360
gtgttcttca aggtggttca gcaaaacagg gagtcgttca atatcaccct ctacgggaga 420
accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 41
<211> 178
<212> PRT
<213> Artificial
<220>
<223> US7 (Abeta)
<400> 41
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Val Ala Gly Asn Lys Ser Leu Arg Glu Asp Lys Asp Pro
35 40 45
Trp Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Gly Phe Gly Thr Lys Lys Cys His Tyr Lys Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Arg
85 90 95
Ile Lys Ser Arg Pro Gly Arg Thr Ser Ala Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Gln Gln
115 120 125
Asn Arg Glu Ser Phe Asn Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 42
<211> 805
<212> DNA
<213> Artificial
<220>
<223> XbaI/HindIII restriction fragment of phNGAL124, which encodes
wild type Lcn2 as fusion with the albumin-binding domain cloned
on pASK75. Reading frame starts at position 22 with atg
<400> 42
tctagataac gagggcaaaa aatgaaaaag acagctatcg cgattgcagt ggctctggct 60
ggcttcgcta ccgtagcgca ggcccaggac tccacctcag acctgatccc agccccacct 120
ctgagcaagg tccctctgca gcagaacttc caggacaacc aattccatgg gaagtggtat 180
gtggtaggtc tcgcagggaa tgcaattctc agagaagaca aagacccgca aaagatgtat 240
gccaccatct atgagctgaa agaagacaag agctacaatg tcacctccgt cctgtttagg 300
aaaaagaagt gtgactactg gatcaggact tttgttccag gttcccagcc aggcgagttc 360
acgctgggca acattaagag ttaccctgga ttaacgagtt acctcgtccg agtggtgagc 420
accaactaca accagcatgc tatggtgttc ttcaagaaag tttctcaaaa cagggagtac 480
ttcaagatca ccctctacgg gagaaccaag gagctgactt cggaactaaa ggagaacttc 540
atccgcttct ccaaatctct gggcctccct gaaaaccaca tcgtcttccc tgtcccaatc 600
gaccagtgta tcgacggcag cgcttggtcc cacccgcagt tcgaaaaata ggcccacctg 660
gctgaagcta aagttctggc taaccgtgaa ctggacaaat acggtgtttc cgactactac 720
aaaaacctca tcaacaacgc taaaaccgtt gaaggtgtta aagctctgat cgacgaaatt 780
ctcgcagcac tgccgtaata agctt 805
<210> 43
<211> 178
<212> PRT
<213> Artificial
<220>
<223> H1-G1 (Abeta)
<400> 43
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Cys Ala Gly Asn Val Leu Leu Arg Glu Asp Lys Asp Pro
35 40 45
Leu Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Gly Phe Asp Asp Lys Lys Cys Leu Tyr Lys Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Arg
85 90 95
Ile Lys Ser Glu Pro Gly Gly Thr Ser Trp Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Glu Val Ala Gln
115 120 125
Asn Arg Glu Thr Phe Asn Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 44
<211> 178
<212> PRT
<213> Homo sapiens
<220>
<221> MISC_FEATURE
<223> wt Lcn2
<400> 44
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro
35 40 45
Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn
85 90 95
Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln
115 120 125
Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 45
<211> 21
<212> DNA
<213> Artificial
<220>
<223> Primer
<400> 45
cccaggactc cacctcagac c 21
<210> 46
<211> 27
<212> DNA
<213> Artificial
<220>
<223> Primer
<400> 46
actgcgggtg ggaccaagcg ctgccgt 27
<210> 47
<211> 58
<212> DNA
<213> Artificial
<220>
<223> degenerate oligonucleotide DH-4
<220>
<221> misc_feature
<222> (39)..(39)
<223> y is c or t
<400> 47
ggacaaccaa ttccatggga agtggtatgt ggtaggtgyt gcagggaatg tgttgctc 58
<210> 48
<211> 18
<212> DNA
<213> Artificial
<220>
<223> Primer J08rev
<400> 48
gctgccgtcg atacactg 18
<210> 49
<211> 534
<212> DNA
<213> Artificial
<220>
<223> DNA sequence for the mature protein H1GA without Strep-tag II.
Reading frame starts with the first nucleotide of the following
sequence
<400> 49
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaag tggtatgtgg taggtgctgc agggaatgtg 120
ttgctcagag aagacaaaga cccgcttaag atgtatgcca ccatctatga gctgaaagaa 180
gacaagagct acaatgtcac cagtgtcggg tttgatgata agaagtgttt gtacaagatc 240
cggacttttg ttccaggttc ccagccaggc gagttcacgc tgggcaggat taagagtgag 300
cctggaggta cgagttggct cgtccgagtg gtgagcacca actacaacca gcatgctatg 360
gtgttcttca aggaggttgc gcaaaacagg gagacgttca atatcaccct ctacgggaga 420
accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 50
<211> 178
<212> PRT
<213> Artificial
<220>
<223> H1GA (Abeta)
<400> 50
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Ala Ala Gly Asn Val Leu Leu Arg Glu Asp Lys Asp Pro
35 40 45
Leu Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Gly Phe Asp Asp Lys Lys Cys Leu Tyr Lys Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Arg
85 90 95
Ile Lys Ser Glu Pro Gly Gly Thr Ser Trp Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Glu Val Ala Gln
115 120 125
Asn Arg Glu Thr Phe Asn Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
<210> 51
<211> 534
<212> DNA
<213> Artificial
<220>
<223> DNA sequence for the mature protein H1GV without Strep-tag II.
Reading frame starts with the first nucleotide of the following
sequence
<400> 51
caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60
aacttccagg acaaccaatt ccatgggaag tggtatgtgg taggtgttgc agggaatgtg 120
ttgctcagag aagacaaaga cccgcttaag atgtatgcca ccatctatga gctgaaagaa 180
gacaagagct acaatgtcac cagtgtcggg tttgatgata agaagtgttt gtacaagatc 240
cggacttttg ttccaggttc ccagccaggc gagttcacgc tgggcaggat taagagtgag 300
cctggaggta cgagttggct cgtccgagtg gtgagcacca actacaacca gcatgctatg 360
gtgttcttca aggaggttgc gcaaaacagg gagacgttca atatcaccct ctacgggaga 420
accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 480
ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534
<210> 52
<211> 178
<212> PRT
<213> Artificial
<220>
<223> H1GV (A beta)
<400> 52
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val
1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30
Val Val Gly Val Ala Gly Asn Val Leu Leu Arg Glu Asp Lys Asp Pro
35 40 45
Leu Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60
Asn Val Thr Ser Val Gly Phe Asp Asp Lys Lys Cys Leu Tyr Lys Ile
65 70 75 80
Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Arg
85 90 95
Ile Lys Ser Glu Pro Gly Gly Thr Ser Trp Leu Val Arg Val Val Ser
100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Glu Val Ala Gln
115 120 125
Asn Arg Glu Thr Phe Asn Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu
130 135 140
Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175
Asp Gly
Claims (54)
- 検出可能な親和性を有する所定の非天然標的に結合することができ、ヒトリポカリン2(Lcn2、hNGAL)に由来する突然変異タンパク質の生成方法であって、
ヒトLcn2タンパク質をコードする核酸分子を、ヒトLcn2の直鎖状ポリペプチド配列における配列位置96、100、及び106に対応する配列位置のうちのいずれかの少なくとも1つをコードするヌクレオチドトリプレットで突然変異誘発させて、1つ又はそれ以上の突然変異タンパク質核酸分子を生じるステップ(a)を備えている方法。 - 請求項1の方法において、
ステップ(a)で得られた1つ以上の突然変異タンパク質核酸分子を適切な発現系で発現させるステップ(b)と、
所定の標的に対して検出可能な結合親和性を有する1つ又はそれ以上の突然変異タンパクを、選択及び/又は単離によって濃縮するステップ(c)とをさらに備えている方法。 - 請求項1又は2の方法において、
前記核酸分子を、前記Lcn2の配列位置96、100、及び106のいずれかをコードするヌクテオチドトリプレットの少なくとも1つ、2つ又は3つ全てで突然変異誘発させることをさらに備えている方法。 - 請求項1~3のいずれか1項の方法において、
前記核酸分子を、hLcn2の直鎖状ポリペプチド配列における配列位置36、40、41、49、52、68、70、72、73、77、79、81、103、125、127、132及び134のいずれかをコードする少なくとも1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16又は17全てのヌクレオチドトリプレットで突然変異誘発させることをさらに備えている方法。 - 請求項1~4のいずれか1項の方法において、
ヌクレオチドトリプレットのサブセットによって置換を可能にすることのみによって、hLcn2の直鎖状ポリペプチド配列における配列位置36、40、41、49、52、68、70、72、73、77、79、81、96、100、103、106、125、127、132及び134のいずれかをコードする少なくとも1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20のヌクレオチドトリプレットでランダム突然変異誘発させることをさらに備えている方法。 - 請求項1~5のいずれか1項の方法において、
システインをコードするヌクレオチドトリプレットは、突然変異誘発時の置換に用いられない方法。 - 請求項1~6のいずれか1項の方法により得られるLcn2タンパク質由来の突然変異タンパク質であって、
Lcn2の直鎖状ポリペプチド配列における配列位置96、100、及び106に対応する配列位置のうちのいずれかにおける少なくとも1つの突然変異したアミノ酸残基を備え、検出可能な親和性を有する所定の非天然標的に結合することができる突然変異タンパク質。 - 請求項7の突然変異タンパク質において、
Lcn2の直鎖状ポリペプチド配列における配列位置96、100、及び106に2つ又は3つの突然変異したアミノ酸残基を備えている突然変異タンパク質。 - 請求項7又は8の突然変異タンパク質において、
hLcn2の直鎖状ポリペプチド配列における配列位置36、40、41、49、52、68、70、72、73、77、79、81、103、125、127、132及び134に対応する配列位置のいずれかで少なくとも1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16又は17の突然変異したアミノ酸残基をさらに備えている突然変異タンパク質。 - 請求項8又は9の突然変異タンパク質において、
Lcn2の直鎖状ポリペプチド配列における配列位置36、40、41、49、52、68、70、72、73、77、79、81、96、100、103、106、125、127、132及び134のいずれかで少なくとも2、3、4、5、6、7、8、9、10、11、12、13、14、15、16又は17の突然変異したアミノ酸残基をさらに備えている突然変異タンパク質。 - 請求項10の突然変異タンパク質において、
Lcn2の直鎖状ポリペプチド配列における配列位置36、40、41、49、52、68、70、72、73、77、79、81、96、100、103、106、125、127、132及び134のいずれかで18、19又は20の突然変異したアミノ酸残基をさらに備えている突然変異タンパク質。 - 請求項7~11のいずれか1項の突然変異タンパク質において、
前記非天然標的は、ペプチド、タンパク質、タンパク質のフラグメント又はドメイン、及び有機小分子からなる群から選択される突然変異タンパク質。 - 請求項12の突然変異タンパク質において、
前記有機小分子は、免疫学的ハプテンの特性を示す化合物である突然変異タンパク質。 - 請求項12の突然変異タンパク質において、
前記ペプチドは、2~45アミノ酸の長さを有する突然変異タンパク質。 - 請求項14の突然変異タンパク質において、
前記ペプチドは、アミロイドベータペプチドである突然変異タンパク質。 - 請求項15の突然変異タンパク質において、
前記アミロイドベータペプチドは、Aβ40ペプチド又はAβ42ペプチドである突然変異タンパク質。 - 請求項15又は16の突然変異タンパク質において、
成熟hLcn2の野生型アミノ酸配列について、Leu36→Val又はCys、Ala40→Tyr又はLys又はVal、Ile41→Thr又はSer又はLeu、Gln49→Leu又はTrp、Leu70→Gly、Arg72→Gly又はAsp、Lys73→Leu又はThr又はAsp、Asp77→Asn又はHis又はLeu、Trp79→Lys、Asn96→Ile又はArg、Tyr100→Gln又はArg又はGlu、Leu103→Met又はArg又はGly、Tyr106→Tyr又はAla又はTrp、Lys125→Thr又はVal又はGlu、Serl27→Gly又はGln又はAla、Tyr132→Met又はSer又はThr、及びLys134→Asnからなる群から選択される、少なくとも3、4、5、6、7、8、9、10、11、又は12のアミノ酸置換を含む突然変異タンパク質。 - 請求項17の突然変異タンパク質において、
配列番号39(S1-A4)又は配列番号41(US7)又は配列番号43(H1-G1)の配列を有する突然変異タンパク質。 - 請求項12の突然変異タンパク質において、
前記タンパク質は、フィブロネクチン又はそのドメインである突然変異タンパク質。 - 請求項19の突然変異タンパク質において、
前記フィブロネクチンドメインは、エキストラドメインB又はそのフラグメントである突然変異タンパク質。 - 請求項19又は20の突然変異タンパク質において、
成熟hLcn2の野生型アミノ酸配列について、Leu36→Lys又はGlu又はArg又はAla、Ala40→His又はMet又はThr又はSer、Ile41→Asp又はArg又はTrp又はLeu、Gln49→Arg又はAla又はTyr、Leu70→Leu又はArg又はMet、Arg72→Val又はArg又はGln又はMet、Lys73→His又はArg又はSer、Asp77→Asn又はHis又はLys又はArg、Trp79→Arg又はMet又はLeu、Asn96→Lys又はAla又はSer、Tyr100→Trp又はPro又はLys、Leu103→His又はPro、Tyr106→Phe又はTrp又はThr、Lys125→Arg又はHis又はThr、Ser127→Thr又はPhe又はAla、Tyr132→Leu又はPhe、Lys134→Glu又はHis又はGly又はPhe、及びSer146→Asnからなる群から選択される、少なくとも3、4、5、6、7、8、9、10、11、又は12のアミノ酸置換を含む突然変異タンパク質。 - 請求項21の突然変異タンパク質において、
配列番号20(N7A)又は配列番号22(N7E)又は配列番号24(N9B)又は配列番号26(N10D)の配列を有する突然変異タンパク質。 - 請求項7~22のいずれか1項の突然変異タンパク質において、
成熟hLcn2の野生型アミノ酸配列について、Glu28→His、及びCys87→Serからなる群から選択されるアミノ酸置換をさらに含む突然変異タンパク質。 - 請求項7~23のいずれか1項の突然変異タンパク質において、
Lcn2の直鎖状ポリペプチド配列における配列位置137及び145に対応する配列位置のいずれかで突然変異したアミノ酸残基を備えていない突然変異タンパク質。 - 請求項7~24のいずれか1項の突然変異タンパク質において、
成熟hLcn2の野生型アミノ酸配列について、Tyr52→Gln又はVal、Ser68→Lys又はAsn、及びArg81→Trp又はAsn又はHisからなる群から選択されるアミノ酸置換を含む突然変異タンパク質。 - 請求項7~25のいずれか1項の突然変異タンパク質において、
有機分子、酵素標識、放射性標識、呈色標識、蛍光標識、発色標識、発光標識、ハプテン、ジゴキシゲニン、ビオチン、細胞増殖抑制剤、毒素、金属錯体、金属及びコロイド金からなる群から選択される化合物に接合された突然変異タンパク質。 - 請求項7~26のいずれか1項の突然変異タンパク質において、
融合パートナーであるタンパク質、タンパク質ドメイン、又はペプチドに、そのN末端及び/又はそのC末端で融合された突然変異タンパク質。 - 請求項7~26のいずれか1項の突然変異タンパク質において、
血清半減期を延長する化合物に接合された突然変異タンパク質。 - 請求項28の突然変異タンパク質において、
前記化合物は、ポリアルキレングリコール分子、ヒドロエチルデンプン、免疫グロブリンのFc部分、免疫グロブリンのCH3ドメイン、免疫グロブリンのCH4ドメイン、アルブミン結合ペプチド、又はアルブミン結合タンパク質からなる群から選択される血清半減期を延長する突然変異タンパク質。 - 請求項29の突然変異タンパク質において、
前記ポリアルキレングリコールは、ポリエチレン(PEG)又はその活性誘導体である突然変異タンパク質。 - 請求項27の突然変異タンパク質において、
前記融合パートナーは、前記突然変異タンパク質の血清半減期を延長するタンパク質ドメインである突然変異タンパク質。 - 請求項31の突然変異タンパク質において、
前記タンパク質ドメインは、免疫グロブリンのFc部分、免疫グロブリンのCH3ドメイン、免疫グロブリンのCH4ドメイン、アルブミン結合ペプチド、又はアルブミン結合タンパク質である突然変異タンパク質。 - 請求項29又は32の突然変異タンパク質において、
前記アルブミン結合タンパク質は、細菌性アルブミンドメイン又はリポカリン突然変異タンパク質である突然変異タンパク質。 - 請求項33の突然変異タンパク質において、
前記細菌性アルブミン結合ドメインは、連鎖球菌性タンパク質Gのアルブミン結合ドメインである突然変異タンパク質。 - 請求項29又は32の突然変異タンパク質において、
前記アルブミン結合ペプチドは、Cys-Xaa1-Xaa2-Xaa3-Xaa4-Cysの式を有し、Xaa1はAsp、Asn、Ser、Thr又はTrpであり、Xaa2はAsn、Gln、His、Ile、Leu又はLysであり、Xaa3はAla、Asp、Phe、Trp又はTyrであり、Xaa4はAsp、Gly、Leu、Phe、Ser又はThrである突然変異タンパク質。 - 請求項1~35のいずれか1項の突然変異タンパク質において、
1μM若しくはそれ以下、100μM若しくはそれ以下、1μM若しくはそれ以下、500nM若しくはそれ以下、200nM若しくはそれ以下、100nM若しくはそれ以下、50nM若しくはそれ以下、10nM若しくはそれ以下、又は1nM若しくはそれ以下のKD値で非天然標的に結合する突然変異タンパク質。 - 請求項7~36のいずれか1項の突然変異タンパク質をコードするヌクレオチド配列を備えている核酸分子。
- 請求項37の核酸分子において、
前記核酸分子の発現を可能にする制御配列に動作可能に結合された核酸分子。 - 請求項37又は38の核酸分子において、
ベクターの中に含まれている核酸分子。 - 請求項37又は38の核酸分子において、
ファージミドベクターの中に含まれている核酸分子。 - 請求項37~40のいずれか1項の核酸分子を含む宿主細胞。
- 請求項7~36のいずれか1項の突然変異タンパク質の産生方法であって、
前記突然変異タンパク質、前記突然変異タンパク質のフラグメント、又は前記突然変異タンパク質と他のポリペプチドとの融合タンパク質は、遺伝子工学的方法によって、前記突然変異タンパク質をコードする核酸から産生される方法。 - 請求項42の方法において、
前記突然変異タンパク質は、細菌性又は真核の宿主生物で産生され、この宿主生物又はその培養物から分離される方法。 - 請求項7~36のいずれか1項の少なくとも1つの突然変異タンパク質を含む医薬組成物。
- 所定の標的を結合して検出するための請求項7~36のいずれか1項の突然変異タンパク質の使用であって、
前記標的を含むと予想される試験サンプルに前記突然変異タンパク質を接合するステップ(a)と、
適切なシグナルによって前記突然変異タンパク質/標的複合体を検出するステップ(b)とを備えている突然変異タンパク質の使用。 - 事前選択された部位を化合物が標的とするための請求項7~36のいずれか1項の突然変異タンパク質の使用であって、
前記化合物に前記突然変異タンパク質を接合するステップ(a)と、
前記事前選択された部位に前記突然変異タンパク質/化合物複合体を送達するステップ(b)とを備えている突然変異タンパク質の使用。 - 所定の標的との複合体形成のための請求項7~36のいずれか1項の突然変異タンパク質の使用。
- 医薬組成物の製造のための請求項7~36のいずれか1項の突然変異タンパク質の使用。
- 請求項15~18のいずれか1項の突然変異タンパク質の使用であって、
前記医薬組成物は、神経変性疾患の治療に用いられる突然変異タンパク質の使用。 - 請求項47の使用において、
前記神経変性疾患は、アルツハイマー病である使用。 - 請求項19~22のいずれか1項の突然変異タンパク質の使用であって、
前記医薬組成物は、癌の治療、線維症の治療、神経変性疾患を含む疾患の治療、又は炎症の治療に用いられる突然変異タンパク質の使用。 - 請求項7~36のいずれか1項の突然変異タンパク質を含む診断又は分析キット。
- 請求項15~18のいずれか1項の突然変異タンパク質の有効量を哺乳類に投与することを含む哺乳類の神経変性疾患の治療方法。
- 請求項17~20のいずれか1項の突然変異タンパク質の有効量を哺乳類に投与することを含む哺乳類の癌、線維症及び炎症からなる群から選択される疾患の治療方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US26709809P | 2009-12-07 | 2009-12-07 | |
US61/267,098 | 2009-12-07 | ||
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