JP6251170B2 - 安定したタンパク質 - Google Patents
安定したタンパク質 Download PDFInfo
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- JP6251170B2 JP6251170B2 JP2014524446A JP2014524446A JP6251170B2 JP 6251170 B2 JP6251170 B2 JP 6251170B2 JP 2014524446 A JP2014524446 A JP 2014524446A JP 2014524446 A JP2014524446 A JP 2014524446A JP 6251170 B2 JP6251170 B2 JP 6251170B2
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- C07K2319/00—Fusion polypeptide
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- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
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Description
a)GPCRのTM1、TM2およびTM3を含むファミリーB Gプロテイン結合型受容体(GPCR)の第1の部分;
b)安定したタンパク質ドメイン;ならびに、
c)GPCRのTM4、TM5、TM6およびTM7を含むGPCRの第2の部分。
1.ドメインは、可溶性で、よく折りたたまれており、1つ以上の発現システムで容易に発現することができる。
2.ドメインのN末端とC末端の間隔は、互い接近しており、通常、5〜17Å、例えば、6〜16Å,7〜15Å,7〜10Å,10〜13Åまたは12〜15Åの範囲である。
3.ドメインは、タンパク質分解の変性と、熱および化学変性に対して耐性がある。
4.ドメインは、さまざまな空間群および結晶パッケージングアレンジメントにおいて高い結晶化能を有する。
(http://products.invitrogen.com/ivgn/product/V79020)
T4LはICL2(左)およびICL3(右)内で示された残基の後ろに挿入された。構築物1aは、T4LがL255とS261の間に挿入されたことを意味する。TMドメインとループのモデルは参考文献[4]に基づいている。
各測定は総細胞材料50μgを用いて2連で実施した。エラーバーは、平均からの標準誤差を示す。
それぞれのケースにおいて、野生型プロファイルを赤で、融合構築物を青で示した。
それぞれのケースにおいて、野生型プロファイルを赤で、融合構築物を青で示した。
GLP1R融合構築物1c内でT4Lと置換されたICL2の位置を、マウス、ラットおよびヒトの他のファミリーB受容体中で強調した。
ファミリーB受容体の内部ループへのT4Lの挿入の影響を調べた。
得られたデータは、ファミリーB受容体がICL3内でのT4L融合を許容できないことを示しているが、ICL2にT4Lを付加すると受容体の生物化学的特性が改善される。
GLP1Rのモデルに従いGLP1Rのループ領域を決定し、ICL2とICL3内の異なった場所にT4LをコードするDNAを挿入した(図1)。蛍光検出サイズ排除クロマトグラフィーを用いて全体の発現と単分散をモニターするためにGLP1R構築物のC末端をEGFPでタグ化した。
標準の分子生物学的技術を用いて、T4リゾチームをヒトGLP1Rの第2および第3の細胞質ループに挿入した。この構築物はHEK293T細胞中で修飾pcDNA3.1から一過的に発現され、そのC末端にEGFPが融合した受容体が生産される。使用説明書に従い、GeneJuice(Merck Biosciences社)を用いて遺伝子導入を実施した。通常、10cmのプレートの接着性細胞3×106個に対して6μgのDNAを遺伝子導入のために用いた。遺伝子導入の40時間前に細胞を採取し、EDTAが全く入っていないプロテアーゼインヒビターカクテル(Roche社)を補完した50mM HEPES pH7.5/150mM NaCl/0.5mM EDTAで再懸濁した。通常、それぞれ650μgのサンプルを1%DDMで総量200μLに4℃で1時間かけて溶解し、次に、50,000rpmで30分間遠心した。50μLの上清を、予めSECバッファー(50mM HEPES pH7.5/150mM NaCl/0.5mM EDTA/0.03%DDM)で平衡化させたBioSEep−SEC−S3000カラム(Phenomenex)にロードし、1mL/分の流速で15分間、流した。その溶離液は、以下の設定のオンライン蛍光光度計に通した:励起波長490nm、蛍光波長513nm、13ゲイン。
[1]Kristiansen,K.Molecular mechanisms of ligand binding,signaling,and regulation within the superFamily of G−protein−coupled receptors:molecular modeling and mutagenesis approaches to receptor structure and function.Pharma & Therap 103,21−80(2004)
[2]Bill RM,Henderson PJ,Iwata S,Kunji ER,Michel H,Neutze R,Newstead S,Poolman B,Tate CG and Vogel H.Overcoming barriers to membrane protein structure determination.Nat Biotechnol.29(4),335−340(2011)
[3]Kobilka BK,Kobilka TS,Daniel K,Regan JW,Caron MG and Lefkowitz RJ.Chimeric alpha 2−,beta 2−adrenergic receptors:delineation of domains involved in effector coupling and ligand binding specificity.Science 240(4857)1310−6(1988)
[4]Frimurer TM and Bywater RP.Structure of the integral membrane domain of the GLP1 receptor.Proteins 35(4),375−86(1999)
[5]Kawate T and Gouaux E.Fluorescence−detection size−exclusion chromatography for precrystallization screening of integral membrane proteins.Structure 14(4),673−81(2004)
Claims (20)
- N末端からC末端にかけて以下を含む融合タンパク質:
a)GPCRのトランスメンブランへリックス(TM)−1、TM−2およびTM−3を含むファミリーB Gプロテイン結合型受容体(GPCR)の第1の部分;
b)GPCRのTM4、TM5、TM6およびTM7を含むGPCRの第2の部分;ならびに、
c)GPCRの第1の部分と第2の部分を結合するGPCRの細胞内ループ2(ICL2)領域に挿入されたT4リゾチームである、安定したタンパク質ドメイン。 - GPCRが、ファミリーB GPCRである請求項1に記載の融合タンパク質。
- ファミリーB GPCRが、グルカゴン様ペプチド1受容体(GLP1R)、グルカゴン様ペプチド2受容体(GLP2R)、カルシトニン受容体(CT)、アミリン/CGRP受容体(AMY1a)、アミリン受容体(AMY2a)、アミリン/CGRP受容体(AMY3a)、CGRP/アデノメデュリン受容体(CGRP1a)、アデノメデュリン/CGRP受容体(AM1a)、アデノメデュリン/CGRP受容体(AM2a受容体)、コルチコトロピン放出因子受容体(CRF1)、ウロコルチン受容体(CRF2)、成長ホルモン放出ホルモン受容体(GHRH)、胃酸分泌抑制ポリペプチド受容体(GIP)、グルカゴン受容体、セクレチン受容体、TIP−39受容体(PTH2)、副甲状腺ホルモン受容体(PTH1)、VIP/PACAP受容体(VPAC1)、PACAP受容体(PAC2)またはVIP/PACAP受容体(VPAC2)である請求項2に記載の融合タンパク質。
- GPCRが、元のGPCRに比べて立体構造の安定性が増加している変異型GPCRである請求項1〜3のいずれか一項に記載の融合タンパク質。
- ヒトGLP1Rのアミノ酸Phe257(配列番号30に示されたアミノ酸配列の第5番目)およびSer261(配列番号30に示されたアミノ酸配列の第9番目)に対応するアミノ酸残基の間の位置において、安定したタンパク質ドメインがGPCRのICL2領域に挿入された請求項1〜4のいずれか一項に記載の融合タンパク質。
- ヒトGLP1Rのアミノ酸Phe257(配列番号30に示されたアミノ酸配列の第5番目)に対応するアミノ酸の後およびアミノ酸Ser261(配列番号30に示されたアミノ酸配列の第9番目)、Phe260(配列番号30に示されたアミノ酸配列の第8番目)またはVal259(配列番号30に示されたアミノ酸配列の第7番目)に対応するアミノ酸の前のGPCRのICL2領域に、安定したタンパク質ドメインが挿入された請求項5に記載の融合タンパク質。
- ヒトGLP1Rのアミノ酸Ser258(配列番号30に示されたアミノ酸配列の第6番目)に対応するアミノ酸の後およびアミノ酸Ser261(配列番号30に示されたアミノ酸配列の第9番目)、Phe260(配列番号30に示されたアミノ酸配列の第8番目)またはVal259(配列番号30に示されたアミノ酸配列の第7番目)に対応するアミノ酸の前のGPCRのICL2領域に、安定したタンパク質ドメインが挿入された請求項5に記載の融合タンパク質。
- さらに、検出可能部分を含む請求項1〜7のいずれか一項に記載の融合タンパク質。
- 検出可能部分が蛍光標識である請求項8に記載の融合タンパク質。
- 検出可能部分が放射性標識である請求項8に記載の融合タンパク質。
- 検出可能部分が酵素標識である請求項8に記載の融合タンパク質。
- 検出可能部分がEGFPである請求項8または9に記載の融合タンパク質。
- 請求項1〜12のいずれか一項に記載の融合タンパク質をコードするポリヌクレオチド。
- 請求項13に記載のポリヌクレオチドを含む宿主細胞。
- 請求項1〜12のいずれか一項に記載の融合タンパク質を含む結晶。
- 可溶化型の態様、他のタンパク質を含まない態様、または、固相担体に固定された態様である請求項1〜12のいずれか一項に記載の融合タンパク質。
- 結晶化のための、リガンド結合スクリーニングもしくはアッセイの開発における創薬のための、または、バイオセンサーとしての請求項1〜12のいずれか一項に記載の融合タンパク質の使用。
- 請求項1〜12のいずれか一項に記載の融合タンパク質の提供とそれを結晶化して結晶を得ることを含む請求項1〜12のいずれか一項に記載の融合タンパク質の結晶化方法。
- 請求項14に記載の宿主細胞宿主細胞を培養し、融合タンパク質を発現してそのタンパク質を単離することによって融合タンパク質を提供する請求項18に記載の方法。
- 脂質キュービック相結晶構造解析を用いて結晶化が行われる請求項18または19に記載の方法。
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US10287349B2 (en) | 2014-10-31 | 2019-05-14 | Abilita Bio, Inc. | Modified membrane spanning proteins and methods for the preparation and use thereof |
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Family Cites Families (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1070257A2 (en) | 1998-04-06 | 2001-01-24 | Lerner Pharmaceuticals, Inc. | Directed evolution biosensors |
US6692696B1 (en) | 1998-06-18 | 2004-02-17 | ARETé ASSOCIATES | Biosensor |
WO2000070343A2 (en) | 1999-05-14 | 2000-11-23 | Repliscent, Inc. | G-protein coupled receptor based biosensors and sense replication systems |
AU2001243145A1 (en) | 2000-02-09 | 2001-08-20 | Human Genome Sciences, Inc. | Human g-protein chemokine receptor (ccr5) hdgnr10 |
US7678539B2 (en) | 2000-08-10 | 2010-03-16 | Corning Incorporated | Arrays of biological membranes and methods and use thereof |
WO2002029050A2 (en) * | 2000-10-06 | 2002-04-11 | Bayer Aktiengesellschaft | Regulation of human secretin receptor-like gpcr |
JP2005503124A (ja) | 2001-02-14 | 2005-02-03 | アムジェン インコーポレイテッド | Gタンパク質共役受容体分子及びその使用 |
US7294472B2 (en) | 2001-03-14 | 2007-11-13 | Caden Biosciences | Method for identifying modulators of G protein coupled receptor signaling |
US20050009204A1 (en) | 2003-07-11 | 2005-01-13 | Ye Fang | Multiplexed binding assays for receptor arrays |
AU2003227268A1 (en) | 2002-03-28 | 2003-10-13 | Takeda Chemical Industries, Ltd. | Novel screening method |
JP2004238384A (ja) | 2002-03-28 | 2004-08-26 | Takeda Chem Ind Ltd | 新規スクリーニング方法 |
US20040096914A1 (en) | 2002-11-20 | 2004-05-20 | Ye Fang | Substrates with stable surface chemistry for biological membrane arrays and methods for fabricating thereof |
JP2006340717A (ja) | 2005-05-11 | 2006-12-21 | Sekisui Chem Co Ltd | Gタンパク質共役型受容体に対する結合性評価方法及び評価用組成物、融合タンパク質、並びに、遺伝子 |
FR2890174B1 (fr) | 2005-08-30 | 2009-04-24 | Cis Bio Internat Sa | Procede pour la mise en evidence d'un processus biologique par mesure d'un fret |
EP3926045A1 (en) * | 2007-03-22 | 2021-12-22 | Heptares Therapeutics Limited | Mutant proteins and methods for selecting them |
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JP5560448B2 (ja) * | 2007-10-22 | 2014-07-30 | ザ スクリプス リサーチ インスティテュート | 膜タンパク質の高分解能結晶を得るための方法および組成物 |
GB0724051D0 (en) | 2007-12-08 | 2008-01-16 | Medical Res Council | Mutant proteins and methods for producing them |
GB0724860D0 (en) | 2007-12-20 | 2008-01-30 | Heptares Therapeutics Ltd | Screening |
GB0802474D0 (en) | 2008-02-11 | 2008-03-19 | Heptares Therapeutics Ltd | Mutant proteins and methods for selecting them |
DE08718604T1 (de) | 2008-03-05 | 2009-12-24 | Medical Research Council | Kristallstruktur |
WO2009114020A1 (en) | 2008-03-14 | 2009-09-17 | Sgl Network, Inc. | Systems and methods for biometric authentication of monetary fund transfer |
GB0910725D0 (en) | 2009-06-22 | 2009-08-05 | Heptares Therapeutics Ltd | Mutant proteins and methods for producing them |
US8900591B2 (en) | 2010-08-20 | 2014-12-02 | Heptares Therapeutics Limited | Vaccines comprising mutant GPCRs with increased conformational stability relative to parent receptors |
WO2012030735A1 (en) * | 2010-08-30 | 2012-03-08 | Confometrx, Inc. | Method and composition for crystallizing a family c gpcr |
US20140031525A1 (en) | 2011-01-21 | 2014-01-30 | Heptares Therapeutics Limited | Mutant g-protein coupled receptor proteins and methods for producing them |
US20120288913A1 (en) | 2011-05-13 | 2012-11-15 | The Scripps Research Institute | Novel fusion partners for the purpose of crystallizing g-protein coupled receptors |
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EP2617732A1 (en) | 2012-01-19 | 2013-07-24 | Vib Vzw | Tools and methods for expression of membrane proteins |
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