WO2000049183A1 - Detection de la camp dans des cellules vivantes par transfert d'energie de resonance de la fluorescence a l'aide de variants de la proteine gfp - Google Patents

Detection de la camp dans des cellules vivantes par transfert d'energie de resonance de la fluorescence a l'aide de variants de la proteine gfp Download PDF

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Publication number
WO2000049183A1
WO2000049183A1 PCT/US2000/004164 US0004164W WO0049183A1 WO 2000049183 A1 WO2000049183 A1 WO 2000049183A1 US 0004164 W US0004164 W US 0004164W WO 0049183 A1 WO0049183 A1 WO 0049183A1
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WO
WIPO (PCT)
Prior art keywords
camp
reporter
energy transfer
construct
resonance energy
Prior art date
Application number
PCT/US2000/004164
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English (en)
Inventor
Xianqiang Li
Yu Fang
Original Assignee
Clontech Laboratories, Inc.
Priority date (The priority date 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 date listed.)
Filing date
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Application filed by Clontech Laboratories, Inc. filed Critical Clontech Laboratories, Inc.
Publication of WO2000049183A1 publication Critical patent/WO2000049183A1/fr

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Classifications

    • 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
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the present invention relates generally to the field of molecular and cellular biology. More specifically, the present invention relates to detection of cyclic AMP using fluorescent reporter construct(s).
  • Fluorescence resonance energy transfer is a process in which an excited fluorophore (the donor) transfers its excited energy to a light absorbing molecule (the acceptor). Fluorescence resonance energy transfer is a non-destructive spectroscopic method that can monitor the proximity and relative angular orientation of fluorophores in living cells.
  • Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jellyfish, Aequorea victoria.
  • the cDNA encoding GFP can be fused with coding sequences from a number of other proteins; such fusion proteins usually fluoresce a s well as retain the biochemical function and cellular localization of the additional protein.
  • GFP, as well as mutants of GFP with shifted wavelengths of excitation or emission can serve as donors an d acceptors for fluorescence resonance energy transfer.
  • CFP Cyan
  • YFP Yellow
  • CFP and YFP contain 6 and 4 mutations, respectively. They are Tyr66Tyr, Phe66Leu, Ser65Thr, Asnl45Ile, Metl53Thr, an d Vall63Ala in CFP and Ser65Gly, Nal68Leu, Ser72Ala, and Thr203Tyr in YFP.
  • Enhanced CFP (ECFP) and enhanced YFP (EYFP) are encoded by genes with human-optimized codons. ECFP is excited at 433 nm and emits at 475 nm. EYFP is excited at 523 or 488 nm and emits at 527 nm.
  • cAMP is an important second messenger in signal transduction pathway.
  • Two regulatory (R) and two catalytic (C) subunits comprise the cAMP-dependent protein kinase. When cAMP binds to the R subunits, the C subunits dissociate and continue to phosphorylate other proteins.
  • C subunits were labeled with fluorescein isothiocyanate (FITC) and R subunits were labeled with tetramethylrhodamine isothiocyanate (Rhodamine).
  • the prior art is deficient in a single fluorescing reporter construct that detects cAMP levels in vivo.
  • the pre sent invention fulfills this long-standing need and desire in the art.
  • the present invention establishes a technology to monitor cAMP changes in living cells using two GFP variants (ECFP and EYFP). Since proteins can be tagged by GFP or one of its mutants and retain functional activity following expression, the present invention establishes a technology to monitor cAMP changes in living cells.
  • the present invention is a n improvement over previous technology because only the R subunit of cAMP-dependent protein kinase, containing two cAMP binding domains, need be labeled.
  • the present invention describes a construct in which the gene encoding the R subunit is essentially a linker between the genes encoding ECFP and EYFP.
  • the R subunit undergoes a conformational change, thereby reducing the distance between ECFP and EYFP and allowing detection by fluorescence resonance energy transfer.
  • One object of the present invention is to provide a single construct by which cAMP levels can be detected readily in vivo .
  • a reporter construct for monitoring cAMP levels comprising: a) a fluorophore; b) linker DNA, comprising one o r more cAMP binding (CAB) sites; and c) a light absorbing molecule.
  • This invention further embodies a recombinant DNA molecule encoding the reporter construct and a kit comprising the construct.
  • a reporter construct for monitoring cAMP levels comprising: a) ECFP; b) linker DNA, comprising two cAMP binding (CAB) sites; and c) EYFP.
  • This embodiment further comprises a recombinant DNA molecule, with a specific embodiment having the sequence shown in SEQ ID No. 1.
  • a method of monitoring cAMP levels in a medium comprising the steps of: a) combining a reporter construct comprising: 1) a fluorophore; 2) linker DNA, comprising one or more cAMP binding (CAB) sites; and 3) a light absorbing molecule, with an acceptable medium to produce reporter- containing medium; b) combining a control construct with th e acceptable medium, thereby producing control-containing medium, wherein the control construct comprises the fluorophore, the light absorbing molecule and the linker DNA absent the cAMP binding (CAB) sites; and c) measuring fluourescence resonance energy transfer (FRET) in said reporter-containing medium an d said control-containing medium, wherein a greater amount of fluourescence resonance energy transfer in said reporter- containing medium than in said control-containing medium indicates a greater amount of cAMP in said reporter-containing medium than in said control-containing medium, wherein a lesser amount of fluourescence resonance energy transfer in said reporter-containing
  • This embodiment may further comprise the steps of: d) contacting th e reporter-containing medium with a stimulus; and e) measuring fluourescence resonance energy transfer in the reporter- containing medium prior to and following contact with the stimulus, wherein a greater amount of fluourescence resonance energy transfer in the reporter-containing medium following contact with the stimulus than prior to contact with the stimulus indicates an induction of cAMP levels in response to the stimulus , wherein a lesser amount of fluourescence resonance energy transfer following contact with the stimulus than prior to contact with the stimulus indicates an inhibition of cAMP levels in response to the stimulus.
  • Figure 1 shows a schematic of the present invention demonstrating in vivo monitoring of cAMP.
  • Figure 2 shows the sequence of pECFP-CAB-EYFP.
  • the present invention establishes a technology to monitor cAMP changes in living cells using two GFP variants, ECFP and EYFP.
  • the present invention is an improvement over the prior art because only the R subunit of cAMP-dependent protein kinase, containing two cAMP binding domains, need be labeled.
  • the present invention describes a construct in which the gene encoding the R subunit is essentially a linker between the genes encoding ECFP and EYFP.
  • the R subunit undergoes a conformational change, thereby reducing the distance between ECFP and EYFP and allowing detection by fluorescence resonance energy transfer.
  • the technology described herein with mutants of GFP is superior over previous reports using fluorescence resonance energy transfer because there are no substrates or enzymatic reaction required. Furthermore, it is useful in in vivo applications because the compounds that induce intracellular levels of cAMP can be administered directly to cells expressing the FRET-cAMP construct, pECFP-CAB-EYFP. This construct allows high throughput screening of drugs involved in cAMP signal transduction pathways .
  • the present invention is directed toward a single, flourescently-labelled reporter construct to detect and monitor cAMP levels in vivo.
  • the present invention is directed towards a reporter construct for monitoring cAMP levels, comprising: a) a fluorophore; b) linker DNA, comprising one or more cAMP binding (CAB) sites; and c) a light absorbing molecule.
  • the flourophore is selected from the group consisting of ECFP and EGFP and the light absorbing molecule is selected from the group consisting of EYFP and EBFP.
  • the present invention further embodies a recombinant DNA molecule encoding the reporter construct and a kit comprising the construct.
  • One embodiment of the present invention is specifically directed toward a reporter construct for monitoring cAMP levels comprising: a) ECFP; b) linker DNA, comprising two cAMP binding (CAB) sites; and c) EYFP.
  • a recombinant DNA molecule comprising this construct would have the sequence shown in SEQ ID No. 1.
  • the present invention is further directed to a method of monitoring cAMP levels in a medium, comprising the steps of: a) combining a reporter construct comprising: 1) a fluorophore; 2 ) linker DNA, comprising one or more cAMP binding (CAB) sites; an d 3) a light absorbing molecule, with an acceptable medium to produce reporter-containing medium; b) combining a control construct with the acceptable medium, thereby producing control- containing medium, wherein the control construct comprises th e fluorophore, the light absorbing molecule and the linker DNA absent the cAMP binding (CAB) sites; and c) measuring fluourescence resonance energy transfer (FRET) in the reporter- containing medium and the control-containing medium.
  • a reporter construct comprising: 1) a fluorophore; 2 ) linker DNA, comprising one or more cAMP binding (CAB) sites; an d 3) a light absorbing molecule, with an acceptable medium to produce reporter-containing medium
  • b)
  • a greater amount of fluourescence resonance energy transfer in th e reporter-containing medium than in the control-containing medium indicates a greater amount of cAMP in the reporter- containing medium than in the control-containing medium, while a lesser amount of fluourescence resonance energy transfer in the reporter-containing medium than in the control medium indicates a lesser amount of cAMP in the reporter-containing medium th an in the control medium.
  • This embodiment of the method of the pre sent invention may further comprise the steps of: d) contacting the reporter-containing medium with a stimulus; and e) measuring fluourescence resonance energy transfer in the reporter- containing medium prior to and following contact with th e stimulus.
  • a greater amount of fluourescence resonance energy transfer in the reporter-containing medium following contact with the stimulus than prior to contact with the stimulus indicates a n induction of cAMP levels in response to the stimulus.
  • a lesser amount of fluourescence resonance energy transfer following contact with the stimulus than prior to contact with th e stimulus indicates an inhibition of cAMP levels in response to th e stimulus.
  • a representative stimulus may include pharmaceutical drugs, known inducers of cAMP or cAMP pathways, known inhibitors of cAMP or cAMP pathways, putative inducers of cAMP or cAMP pathways or putative inhibitors of cAMP or cAMP pathways.
  • fluorescence resonance energy transfer m a y be measured by CCD cameras, FACS or by fluorometry.
  • reporter refers to a molecule (usually a protein) that is expressed in response to or as a result of a particular biological or molecular event.
  • fluorophore refers to th e fluroescent group in a molecule.
  • the term "light absorbing molecule” refers to the fluorophore molecule which accepts energy from a donor fluorophore.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of th e molecule, and does not limit it to any particular tertiary forms . Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses , plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) th at functions as an autonomous unit of DNA replication in vivo ; i.e., capable of replication under its own control.
  • An “origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "operably linked" and “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host.
  • the expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • the transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • a "cDNA” is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript.
  • An “exon” is an expressed sequence transcribed from the gene locus, whereas an “intron” is a non-expressed sequence that is from th e gene locus.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers , polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "cis-element” is a nucleotide sequence, also termed a “consensus sequence” o r "motif", that interacts with other proteins which can upregulate or downregulate expression of a specicif gene locus.
  • a “signal sequence” can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes .
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription a t levels detectable above background.
  • Within the promoter sequence will be found a transcription initiation site, as well a s protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters often, b u t not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the - 1 0 and -35 consensus sequences.
  • oligonucleotide is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of th e oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in th e presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must b e sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use the method.
  • the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • Recombinant DNA technology refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include “gene splicing",
  • a cell has been "transformed” or “transfected” with exogenous or heterologous DNA when such DNA has b een introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • I n prokaryotes, yeast, and mammalian cells for example, th e transforming DNA may be maintained on an episomal element such as a vector or plasmid.
  • a stably transformed cell is one in which the transforming DNA h as become integrated into a chromosome so that it is inherited b y daughter cells through chromosome replication.
  • a "clone” is a population of cells derived from a single cell or ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed "transgenic".
  • the term "host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant an d animal cells.
  • a recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art.
  • Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis.
  • Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells, and more preferentially, plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.
  • a "heterologous" region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • th e gene when the heterologous region encodes a mammalian gene, th e gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • the coding sequence is a construct where th e coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • a standard Northern blot assay can be used to ascertain the relative amounts of mRNA in a cell or tissue obtained from plant or other transgenic tissue, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art.
  • a standard Southern blot assay may be used to confirm the presence and th e copy number of the gene in transgenic systems, in accordance with conventional Southern hybridization techniques known to those of ordinary skill in the art.
  • Both the Northern blot and Southern blot use a hybridization probe, e.g.
  • radiolabelled cDNA either containing the full-length, single stranded DNA or a fragment of the DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 1 00 consecutive nucleotides in length).
  • the DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art.
  • CAB cAMP binding domains
  • the pECFP-CAB-EYFP construct was then transfected into 293 cells with a CaP Expression Kit (CLONTECH). Expression of both the cyan and yellow colors were detected with similar intensity under a fluorescent microscope.
  • pECFP-CAB-EYFP was transfected into 293 cells. After 1 day, the cells are treated with Forskolin to induce cAMP. Following high affinity cAMP binding to the CAB of the recombinantly expressed R subunit, the R subunit underwent a conformational change reducing the distance between ECFP an d EYFP and allowing detection of fluorescence resonance energy transfer.
  • the plasmid without the cAMP binding sites (pECFP- EYFP) was used as a control. Constructs containing different numbers of cAMP binding sites, thereby resulting in different levels of fluorescence, can be constructed.

Abstract

La présente invention concerne un procédé permettant de surveiller les modifications de la cAMP dans des cellules vivantes grâce à deux variants de la protéine GFP (la ECFP et la EYFP). L'invention se rapporte à un produit de synthèse dans lequel le gène codant la sous-unité R est essentiellement un lieur entre les gènes codant la ECFP et la EYFP. Après que la cAMP se soit liée aux deux domaines de liaison cAMP de la sous-unité R exprimée de façon recombinée, la sous-unité R subit un changement de conformation qui réduit la distance entre la ECFP et la EYFP, changement qui est ensuite détecté par transfert d'énergie de résonance de la fluorescence.
PCT/US2000/004164 1999-02-17 2000-02-17 Detection de la camp dans des cellules vivantes par transfert d'energie de resonance de la fluorescence a l'aide de variants de la proteine gfp WO2000049183A1 (fr)

Applications Claiming Priority (2)

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US12047499P 1999-02-17 1999-02-17
US60/120,474 1999-02-17

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WO2000049183A1 true WO2000049183A1 (fr) 2000-08-24

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075332A2 (fr) * 1999-06-04 2000-12-14 Rmf Dictagene S.A. Utilisation de la sous-unite de regulation de la proteine kinase (pka) dependante de l'ampc obtenue a partir de dictyostelium pour mesurer l'ampc
WO2002090987A2 (fr) * 2001-05-10 2002-11-14 Isis Innovation Limited Marqueurs fluorescents universels
GB2375538A (en) * 2001-02-15 2002-11-20 Glaxo Group Ltd Polypeptide constructs for FRET analysis
WO2003025220A3 (fr) * 2001-09-18 2003-12-11 Carnegie Inst Of Washington Proteines hybrides destinees a la detection d'analytes
EP1435519A1 (fr) * 1997-04-07 2004-07-07 BioImage A/S Une méthode de criblage de substances pour leurs effets sur la concentration cAMP basé sur la translocation intracellulaire de PKA
EP1536020A1 (fr) * 2003-11-26 2005-06-01 Bayerische Julius-Maximilians-Universität Würzburg Dispositifs et procédés pour la determination optique de cAMP in vitro et in vivo
US7777016B2 (en) 2004-10-14 2010-08-17 Carnegie Institution Of Washington Neurotransmitter sensors and methods of using the same
US8173863B2 (en) 2005-10-14 2012-05-08 Carnegie Institution Of Washington Sucrose biosensors and methods of using the same
US8357505B2 (en) 2005-03-04 2013-01-22 Carnegie Institution Of Washington Environmentally stable sensors and methods of using the same
US8530633B2 (en) 2004-10-14 2013-09-10 Carnegie Institution Of Washington Development of sensitive FRET sensors and methods of using the same
CN103376250A (zh) * 2013-07-26 2013-10-30 福州市传染病医院 一种特异性快速检测环腺苷酸的试剂盒及检测方法
US8846365B2 (en) 2005-10-14 2014-09-30 Carnegie Institution Of Washington Nucleic acids encoding phosphate fluorescent indicators and methods of using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ADAMS S. R.: "Fluorescence ratio imaging of cyclic AMP in single cells", LETTERS TO NATURE, vol. 349, 21 February 1997 (1997-02-21), pages 694 - 697, XP002929494 *
MIYAWAKI A.,: "Fluorescent indicators fo Ca+ based on green fluorescent proteins and calmodulin", LETTERS TO NATURE, vol. 388, 28 August 1997 (1997-08-28), pages 882 - 887, XP002058386 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8058008B2 (en) 1997-04-07 2011-11-15 Fisher Bioimage Aps Method for extracting quantitative information relating to an influence on a cellular response
EP1435519A1 (fr) * 1997-04-07 2004-07-07 BioImage A/S Une méthode de criblage de substances pour leurs effets sur la concentration cAMP basé sur la translocation intracellulaire de PKA
WO2000075332A2 (fr) * 1999-06-04 2000-12-14 Rmf Dictagene S.A. Utilisation de la sous-unite de regulation de la proteine kinase (pka) dependante de l'ampc obtenue a partir de dictyostelium pour mesurer l'ampc
WO2000075332A3 (fr) * 1999-06-04 2001-05-03 Rmf Dictagene Sa Utilisation de la sous-unite de regulation de la proteine kinase (pka) dependante de l'ampc obtenue a partir de dictyostelium pour mesurer l'ampc
US6573059B1 (en) 1999-06-04 2003-06-03 Rmf Dictagene S.A. Use of the regulatory subunit of the camp dependent protein kinase (PKA) from dictyostelium for camp measurements
GB2375538A (en) * 2001-02-15 2002-11-20 Glaxo Group Ltd Polypeptide constructs for FRET analysis
WO2002090987A2 (fr) * 2001-05-10 2002-11-14 Isis Innovation Limited Marqueurs fluorescents universels
WO2002090987A3 (fr) * 2001-05-10 2003-06-12 Isis Innovation Marqueurs fluorescents universels
WO2003025220A3 (fr) * 2001-09-18 2003-12-11 Carnegie Inst Of Washington Proteines hybrides destinees a la detection d'analytes
EP2284195A1 (fr) * 2001-09-18 2011-02-16 Carnegie Institution Of Washington Protéines hybrides destinées à la détection d'analytes
WO2005052186A1 (fr) * 2003-11-26 2005-06-09 Bayerische Julius-Maximilians-Universität Würzburg Moyens et procedes permettant de mesurer camp in vitro et in vivo
EP1536020A1 (fr) * 2003-11-26 2005-06-01 Bayerische Julius-Maximilians-Universität Würzburg Dispositifs et procédés pour la determination optique de cAMP in vitro et in vivo
US8889425B2 (en) 2003-11-26 2014-11-18 Bayerische Julius-Maximilians-Universität Würzburg Means and methods for the determination of camp in vitro and in vivo
US7777016B2 (en) 2004-10-14 2010-08-17 Carnegie Institution Of Washington Neurotransmitter sensors and methods of using the same
US8354250B2 (en) 2004-10-14 2013-01-15 Carnegie Institution Of Washington Neurotransmitter sensors and methods of using the same
US8530633B2 (en) 2004-10-14 2013-09-10 Carnegie Institution Of Washington Development of sensitive FRET sensors and methods of using the same
US8357505B2 (en) 2005-03-04 2013-01-22 Carnegie Institution Of Washington Environmentally stable sensors and methods of using the same
US8846365B2 (en) 2005-10-14 2014-09-30 Carnegie Institution Of Washington Nucleic acids encoding phosphate fluorescent indicators and methods of using the same
US8173863B2 (en) 2005-10-14 2012-05-08 Carnegie Institution Of Washington Sucrose biosensors and methods of using the same
CN103376250A (zh) * 2013-07-26 2013-10-30 福州市传染病医院 一种特异性快速检测环腺苷酸的试剂盒及检测方法

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