DK2856161T3 - Assays - Google Patents
Assays Download PDFInfo
- Publication number
- DK2856161T3 DK2856161T3 DK13734141.8T DK13734141T DK2856161T3 DK 2856161 T3 DK2856161 T3 DK 2856161T3 DK 13734141 T DK13734141 T DK 13734141T DK 2856161 T3 DK2856161 T3 DK 2856161T3
- Authority
- DK
- Denmark
- Prior art keywords
- membrane protein
- label
- donor
- acceptor
- population
- Prior art date
Links
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2458/00—Labels used in chemical analysis of biological material
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Claims (15)
1. Assay til vurdering af den konformationelle stabilitet af et membranprotein, omfattende: (a) at tilvejebringe en prøve omfattende en første population og en anden population af et membranprotein; hvor membranproteinet i den første population er mærket med et donormærke, og membranproteinet i den anden population er mærket med et acceptormærke, eller membranproteinet i den første population er mærket med et acceptormærke, og membranproteinet i den anden population er mærket med et donormærke, og hvor populationerne af membranprotein er tilvejebragt i en solubiliseret form, i hvilken membranproteinerne bibeholder strukturel integritet og er i en funktionel form, (b) at udsætte den første og anden population af membranproteinet for et denatureringsmiddel eller en denatureringsbetingelse, (c) og at vurdere aggregation mellem membranproteiner af den første og anden population ved at aktivere donormærket til muliggørelse af en afstandsafhængig interaktion med acceptormærket, hvilken interaktion frembringer et de-tekterbart signal.
2. Assay ifølge krav 1, hvor den første population og anden population af membranproteinet er til stede i prøven i et forhold på 1:1.
3. Assay ifølge krav 1 eller 2, hvor donormærket er kovalent bundet til membranproteinet, og acceptormærket er kovalent bundet til membranproteinet.
4. Assay til vurdering af den konformationelle stabilitet af et membranprotein, omfattende: (a) at tilvejebringe en prøve omfattende en membranproteinpopulation, hvor populationen af membranprotein er tilvejebragt i en solubiliseret form, i hvilken membranproteinerne bibeholder strukturel integritet og er i en funktionel form, (b) at udsætte membranproteinpopulationen for et denatureringsmiddel eller en denatureringsbetingelse, (c) at mærke en af N-terminus eller C-terminus af membranproteinet med et donormærke og den anden af N-terminus eller C-terminus af membranproteinet med et acceptormærke, (d) og at vurdere aggregation af membranproteinerne i populationen ved at aktivere donormærket til muliggørelse af en afstandsafhængig interaktion med acceptormærket, hvilken interaktion frembringer et detekterbart signal.
5. Assay ifølge et hvilket som helst af kravene 1-4, hvor interaktionen mellem donormærket og acceptormærket indebærer overførsel af energi fra et donor-fluorphor til et acceptorfluorphor, eventuelt hvor nævnte donorfluorphor er en lanthanid, såsom Terbium; og/eller hvor nævnte acceptorfluorphor er EGFP eller d2.
6. Assay ifølge et hvilket som helst af kravene 1-5, hvor interaktionen mellem donormærket og acceptormærket er en chemiluminescens-reaktion, eventuelt hvor interaktionen mellem donormærket og acceptormærket indebærer dannelse af singlet-oxygenmolekyler, der udløser en chemiluminescens-reaktion.
7. Assay ifølge et hvilket som helst af kravene 1-6, hvor donormærket og/eller acceptormærket er direkte bundet til membranproteinet, eller hvor donormærket og/eller acceptormærket er indirekte bundet til membranproteinet.
8. Assay ifølge et hvilket som helst af kravene 1-7, hvor prøven tilvejebragt i trin (a) omfatter et eller flere detergenter udvalgt blandt dodecylmaltosid (DDM); Cii-, C10-, Cg- eller Cs-maltosid eller glucosid; lauryldimethylaminoxid (LDAO); og natriumdodecylsulfat (SDS).
9. Assay ifølge et hvilket som helst af kravene 1-8, hvor denatureringsmidlet eller denatureringsbetingelsen er udvalgt blandt en eller flere af varme, et detergent, et kaotropisk middel eller pH.
10. Assay ifølge et hvilket som helst af kravene 1-9, hvor membranproteinet er en G-proteinkoblet receptor (GPCR), eventuelt hvor prøven tilvejebragt i trin (a) omfatter en GPCR-ligand, hvor liganden er en, der binder til en GPCR, når GPCR'en befinder sig i en særlig konformation, eventuelt hvor GPCR-liganden er fra agonistklassen af ligander, og den særlige konformation er en agonist-konformation, eller GPCR-liganden er fra antagonistklassen af ligander, og den særlige konformation er en antagonistkonformation.
11. Assay ifølge et hvilket som helst af de foregående krav, hvor assayet anvendes ved lægemiddelscreening.
12. Fremgangsmåde til udvælgelse af et membranprotein med øget konforma-tionel stabilitet, omfattende: (a) at sammenligne den konformationelle stabilitet af en eller flere mutanter af et moder-membranprotein med den konformationelle stabilitet af moder-mem-branproteinet i henhold til assayet ifølge et hvilket som helst af kravene 1-11 og (b) at udvælge en eller flere mutanter, der har øget konformationel stabilitet i forhold til moder-membranproteinet, eventuelt hvor membranproteinet er en GPCR.
13. Fremgangsmåde ifølge krav 12, omfattende: (a) at tilvejebringe en eller flere mutanter af et moder-membranprotein; (b) at vurdere den konformationelle stabilitet af den ene eller de flere mutanter af moder-membranproteinet ifølge et hvilket som helst af kravene 1-11; (c) at vurdere den konformationelle stabilitet af moder-membranproteinet i henhold til assayet ifølge et hvilket som helst af kravene 1-11; og (d) at udvælge en eller flere mutanter af moder-membranproteinet, som har øget konformationel stabilitet sammenlignet med den konformationelle stabilitet af moderproteinet.
14. Fremgangsmåde ifølge krav 12 eller 13, hvor membranproteinet har øget stabilitet over for en hvilken som helst af varme, et detergent, et kaotropisk middel eller en ekstrem pFI-værdi.
15. Fremgangsmåde til fremstilling af en mutant-GPCR, hvilken fremgangsmåde omfatter: (a) at udføre fremgangsmåden ifølge et hvilket som helst af kravene 12-14, (b) at identificere positionen eller positionerne af den eller de muterede ami-nosyrerester i mutantmembranproteinet eller -membranproteinerne, som er blevet udvalgt på grund af øget stabilitet, og (c) at syntetisere et mutantmembranprotein, som indeholder en erstatnings-aminosyre ved en eller flere af de identificerede positioner.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201261654265P | 2012-06-01 | 2012-06-01 | |
| PCT/GB2013/051464 WO2013179062A2 (en) | 2012-06-01 | 2013-05-31 | Assays |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DK2856161T3 true DK2856161T3 (da) | 2018-07-23 |
Family
ID=48746572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK13734141.8T DK2856161T3 (da) | 2012-06-01 | 2013-05-31 | Assays |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US10458993B2 (da) |
| EP (1) | EP2856161B1 (da) |
| JP (1) | JP6251252B2 (da) |
| CN (1) | CN104350383B (da) |
| DK (1) | DK2856161T3 (da) |
| ES (1) | ES2676827T3 (da) |
| WO (1) | WO2013179062A2 (da) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008114020A2 (en) | 2007-03-22 | 2008-09-25 | Heptares Therapeutics Limited | Mutant g-protein coupled receptors and methods for selecting them |
| GB0724860D0 (en) | 2007-12-20 | 2008-01-30 | Heptares Therapeutics Ltd | Screening |
| DK2742066T3 (da) | 2011-08-10 | 2018-03-05 | Heptares Therapeutics Ltd | Stabile proteiner |
| JP6473366B2 (ja) * | 2015-03-31 | 2019-02-20 | シスメックス株式会社 | 温度判定方法、標的ペプチドの検出方法および温度判定試薬 |
| KR101862198B1 (ko) * | 2016-09-30 | 2018-05-30 | (주)스파크바이오파마 | 2차원 겔 전기영동에서의 열 안정성 변화-기반 형광 차이를 이용한 표적 단백질의 규명 방법 |
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 |
| CN1227265C (zh) | 2000-02-09 | 2005-11-16 | 人类基因组科学公司 | 抗ccr5的抗体 |
| US7678539B2 (en) | 2000-08-10 | 2010-03-16 | Corning Incorporated | Arrays of biological membranes and methods and use thereof |
| US20020048811A1 (en) * | 2000-10-16 | 2002-04-25 | Devreotes Peter N. | Receptor mediated activation of heterotrimeric G-proteins |
| WO2002083736A2 (en) | 2001-02-14 | 2002-10-24 | Amgen, Inc. | G-protein coupled receptor molecules and uses thereof |
| 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 |
| US20060073516A1 (en) | 2002-03-28 | 2006-04-06 | Yasuaki Ito | 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 |
| US7803559B1 (en) | 2003-08-08 | 2010-09-28 | The Regents Of The University Of Ca | Protein aggregation regulators |
| 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 |
| WO2008114020A2 (en) | 2007-03-22 | 2008-09-25 | Heptares Therapeutics Limited | Mutant g-protein coupled receptors and methods for selecting them |
| EP2220245B1 (en) | 2007-10-17 | 2015-04-22 | The Board of Trustees of The Leland Stanford Junior University | Method and composition for crystallizing g protein-coupled receptors |
| EP2203475B1 (en) | 2007-10-22 | 2014-04-16 | The Scripps Research Institute | Methods and compostions for obtaining high-resolution crystals of membrane proteins |
| 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 |
| US20110112037A1 (en) | 2008-03-05 | 2011-05-12 | Heptares Therapeutics Limited BioPark | Crystal structure |
| GB0910725D0 (en) | 2009-06-22 | 2009-08-05 | Heptares Therapeutics Ltd | Mutant proteins and methods for producing them |
| US20130122525A1 (en) * | 2010-05-13 | 2013-05-16 | Cornell University | smFRET WITH MEMBRANE PROTEINS |
| EP3333185A1 (en) | 2010-08-20 | 2018-06-13 | Heptares Therapeutics Limited | Mutant gpcrs |
| US8470561B2 (en) | 2010-08-30 | 2013-06-25 | ConfometRX Inc. | GPCR comprising an IC2 insertion |
| US20140031525A1 (en) | 2011-01-21 | 2014-01-30 | Heptares Therapeutics Limited | Mutant g-protein coupled receptor proteins and methods for producing them |
| CA2832739A1 (en) | 2011-05-13 | 2012-11-22 | Receptos, Inc. | Novel fusion partners for the purpose of crystallizing g-protein coupled receptors |
| DK2742066T3 (da) | 2011-08-10 | 2018-03-05 | Heptares Therapeutics Ltd | Stabile proteiner |
| EP2617732A1 (en) * | 2012-01-19 | 2013-07-24 | Vib Vzw | Tools and methods for expression of membrane proteins |
| US20150261911A1 (en) | 2014-03-13 | 2015-09-17 | Heptares Therapeutics Limited | Crystal structure |
-
2013
- 2013-05-31 ES ES13734141.8T patent/ES2676827T3/es active Active
- 2013-05-31 JP JP2015514599A patent/JP6251252B2/ja active Active
- 2013-05-31 DK DK13734141.8T patent/DK2856161T3/da active
- 2013-05-31 EP EP13734141.8A patent/EP2856161B1/en active Active
- 2013-05-31 US US14/404,768 patent/US10458993B2/en active Active
- 2013-05-31 WO PCT/GB2013/051464 patent/WO2013179062A2/en active Application Filing
- 2013-05-31 CN CN201380028866.7A patent/CN104350383B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP6251252B2 (ja) | 2017-12-20 |
| CN104350383A (zh) | 2015-02-11 |
| JP2015519569A (ja) | 2015-07-09 |
| US10458993B2 (en) | 2019-10-29 |
| EP2856161A2 (en) | 2015-04-08 |
| CN104350383B (zh) | 2017-03-08 |
| EP2856161B1 (en) | 2018-04-18 |
| US20150147822A1 (en) | 2015-05-28 |
| WO2013179062A2 (en) | 2013-12-05 |
| WO2013179062A3 (en) | 2014-03-06 |
| ES2676827T3 (es) | 2018-07-25 |
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