ES2576747T3 - Ensayo de transferencia de energía de resonancia con un resto de sustrato de sinaptobrevina - Google Patents
Ensayo de transferencia de energía de resonancia con un resto de sustrato de sinaptobrevina Download PDFInfo
- Publication number
- ES2576747T3 ES2576747T3 ES10824166.2T ES10824166T ES2576747T3 ES 2576747 T3 ES2576747 T3 ES 2576747T3 ES 10824166 T ES10824166 T ES 10824166T ES 2576747 T3 ES2576747 T3 ES 2576747T3
- Authority
- ES
- Spain
- Prior art keywords
- protease
- synaptobrevin
- energy transfer
- resonance energy
- bont protease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010005730 R-SNARE Proteins Proteins 0.000 title 1
- 102000002215 Synaptobrevin Human genes 0.000 title 1
- 238000003556 assay Methods 0.000 title 1
- 238000002165 resonance energy transfer Methods 0.000 title 1
- 239000000758 substrate Substances 0.000 title 1
- 108030001720 Bontoxilysin Proteins 0.000 abstract 5
- 102000004169 proteins and genes Human genes 0.000 abstract 3
- 108090000623 proteins and genes Proteins 0.000 abstract 3
- 102000034287 fluorescent proteins Human genes 0.000 abstract 2
- 108091006047 fluorescent proteins Proteins 0.000 abstract 2
- 150000002632 lipids Chemical class 0.000 abstract 2
- 239000012528 membrane Substances 0.000 abstract 2
- 108091005804 Peptidases Proteins 0.000 abstract 1
- 239000004365 Protease Substances 0.000 abstract 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 238000003776 cleavage reaction Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 abstract 1
- 230000007017 scission Effects 0.000 abstract 1
- 238000010396 two-hybrid screening Methods 0.000 abstract 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
-
- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96486—Metalloendopeptidases (3.4.24)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Procedimiento basado en células de medición de la actividad proteasa de una proteasa BoNT, que comprende: proporcionar una célula transfectada que produce (I) una proteína híbrida que tiene una estructura de A-BC- D o (II) dos proteínas híbridas que tienen una estructura de A-C-B y A-C-D, respectivamente; en la que A es un dominio transmembrana que no es escindible por la proteasa BoNT y que inserta a través de una membrana lipídica de forma tal que B, C y D se encuentran en el mismo lado de la membrana lipídica, B es una primera proteína fluorescente, C es una secuencia de reconocimiento y escisión de la proteasa BoNT y D es una segunda proteína fluorescente; poner en contacto la célula transfectada con una proteasa BoNT en condiciones que permiten a la célula tomar la proteasa BoNT; y medir la fluorescencia de al menos una de las primera y segunda proteínas fluorescentes en la célula transfectada.
Description
Claims (1)
-
imagen1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US25231509P | 2009-10-16 | 2009-10-16 | |
| US252315P | 2009-10-16 | ||
| PCT/US2010/052847 WO2011047265A1 (en) | 2009-10-16 | 2010-10-15 | Resonance energy transfer assay with synaptobrevin substrate moiety |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2576747T3 true ES2576747T3 (es) | 2016-07-11 |
Family
ID=43876579
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES16159320.7T Active ES2676277T3 (es) | 2009-10-16 | 2010-10-15 | Células modificadas genéticamente para el ensayo de transferencia de energía por resonancia con el resto del sustrato de sinaptobrevina |
| ES10824166.2T Active ES2576747T3 (es) | 2009-10-16 | 2010-10-15 | Ensayo de transferencia de energía de resonancia con un resto de sustrato de sinaptobrevina |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES16159320.7T Active ES2676277T3 (es) | 2009-10-16 | 2010-10-15 | Células modificadas genéticamente para el ensayo de transferencia de energía por resonancia con el resto del sustrato de sinaptobrevina |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US9453254B2 (es) |
| EP (2) | EP3067696B1 (es) |
| DK (1) | DK2488868T3 (es) |
| ES (2) | ES2676277T3 (es) |
| LT (1) | LT3067696T (es) |
| PL (1) | PL2488868T3 (es) |
| WO (1) | WO2011047265A1 (es) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009035476A1 (en) | 2007-09-14 | 2009-03-19 | Biosentinel, Llc | Resonance energy transfer assay with cleavage sequence and spacer |
| US10246492B2 (en) * | 2009-10-16 | 2019-04-02 | Biomadison, Inc. | Botulinum assay with synaptobrevin substrate moiety |
| WO2011090159A1 (ja) * | 2010-01-22 | 2011-07-28 | 独立行政法人科学技術振興機構 | 蛋白質の分解活性を測定するためのプローブ試薬 |
| WO2014079495A1 (en) | 2012-11-21 | 2014-05-30 | Syntaxin Limited | Methods for the manufacture of proteolytically processed polypeptides |
| AU2014301116B2 (en) | 2013-06-28 | 2019-11-14 | Merz Pharma Gmbh & Co. Kgaa | Means and methods for the determination of the biological activity of Neurotoxin polypeptides in cells |
| WO2015061738A1 (en) * | 2013-10-25 | 2015-04-30 | Biomadison, Inc. | Enzyme assay with duplicate fluorophores |
| AU2015301737B2 (en) * | 2014-08-12 | 2021-01-28 | Biomadison, Inc. | Botulinum neurotoxins with modified light chain specifity and methods for producing same |
| WO2017201079A1 (en) | 2016-05-16 | 2017-11-23 | Biomadison, Inc. | Improved assay with synaptobrevin based moiety |
| US12019066B2 (en) * | 2016-05-16 | 2024-06-25 | Biomadison, Inc. | Assay with synaptobrevin based moiety |
| GB201702500D0 (en) | 2017-02-16 | 2017-04-05 | Univ Sheffield | Stable vamp reporter assay |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998036081A2 (en) | 1997-02-13 | 1998-08-20 | Memorial Sloan-Kettering Cancer Center | Hybrid molecules for optically detecting changes in cellular microenvironments |
| US8022172B2 (en) * | 2001-08-28 | 2011-09-20 | Allergan, Inc. | Luminescence resonance energy transfer (LRET) assays for clostridial toxin activity |
| US7332567B2 (en) * | 2001-08-28 | 2008-02-19 | Allergan, Inc. | Fret protease assays for clostridial toxins |
| WO2005076785A2 (en) | 2003-12-19 | 2005-08-25 | Wisconsin Alumni Research Foundation | Method and compositions for detecting botulinum neurotoxin |
| EP2293064B1 (en) * | 2005-04-05 | 2012-04-04 | Allergan, Inc. | Lipophilic dye-based FRET assays for clostridial toxin activity |
| WO2009035476A1 (en) * | 2007-09-14 | 2009-03-19 | Biosentinel, Llc | Resonance energy transfer assay with cleavage sequence and spacer |
-
2010
- 2010-10-15 ES ES16159320.7T patent/ES2676277T3/es active Active
- 2010-10-15 US US13/502,357 patent/US9453254B2/en active Active
- 2010-10-15 LT LTEP16159320.7T patent/LT3067696T/lt unknown
- 2010-10-15 EP EP16159320.7A patent/EP3067696B1/en active Active
- 2010-10-15 EP EP10824166.2A patent/EP2488868B1/en active Active
- 2010-10-15 PL PL10824166.2T patent/PL2488868T3/pl unknown
- 2010-10-15 DK DK10824166.2T patent/DK2488868T3/en active
- 2010-10-15 WO PCT/US2010/052847 patent/WO2011047265A1/en not_active Ceased
- 2010-10-15 ES ES10824166.2T patent/ES2576747T3/es active Active
Also Published As
| Publication number | Publication date |
|---|---|
| WO2011047265A4 (en) | 2011-06-16 |
| LT3067696T (lt) | 2018-09-10 |
| EP3067696A1 (en) | 2016-09-14 |
| PL2488868T3 (pl) | 2016-09-30 |
| EP3067696B1 (en) | 2018-06-06 |
| EP2488868A1 (en) | 2012-08-22 |
| US9453254B2 (en) | 2016-09-27 |
| US20120322092A1 (en) | 2012-12-20 |
| WO2011047265A1 (en) | 2011-04-21 |
| DK2488868T3 (en) | 2016-05-09 |
| EP2488868A4 (en) | 2013-10-02 |
| EP2488868B1 (en) | 2016-03-30 |
| ES2676277T3 (es) | 2018-07-18 |
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