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EP1102791A1 - Proteine a fluorescence verte modifiee - Google Patents

Proteine a fluorescence verte modifiee

Info

Publication number
EP1102791A1
EP1102791A1 EP99940293A EP99940293A EP1102791A1 EP 1102791 A1 EP1102791 A1 EP 1102791A1 EP 99940293 A EP99940293 A EP 99940293A EP 99940293 A EP99940293 A EP 99940293A EP 1102791 A1 EP1102791 A1 EP 1102791A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
replaced
cells
dna
gfp
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.)
Withdrawn
Application number
EP99940293A
Other languages
German (de)
English (en)
Inventor
Philippe Bastiaens
Rainer Pepperkok
Stefan Geley
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cancer Research Technology Ltd
Original Assignee
Imperial Cancer Research Technology Ltd
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
Publication date
Priority claimed from GBGB9817229.9A external-priority patent/GB9817229D0/en
Priority claimed from GBGB9817227.3A external-priority patent/GB9817227D0/en
Priority claimed from GBGB9817225.7A external-priority patent/GB9817225D0/en
Application filed by Imperial Cancer Research Technology Ltd filed Critical Imperial Cancer Research Technology Ltd
Publication of EP1102791A1 publication Critical patent/EP1102791A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • 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
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence

Definitions

  • the present invention relates to fluorescent proteins, in particular to "green fluorescent proteins” (GFPs) and mutants thereof which have altered spectral properties compared to the wild type GFP.
  • GFPs green fluorescent proteins
  • Fluorescent assays for biological systems have been used for several years since, compared to many biological techniques, they have the advantage of, in some circumstances, being able to be carried out non-invasively and also they are able to give real time analysis of particular reactions in complex biological systems in which many reactions are being carried out simultaneously.
  • fluorescent assays In conjunction with the development of physical techniques for fluorescent assays has been the development of biological reporter constructs which serve as monitors of reactions, for example, within a cell.
  • fluorescent proteins that do not require cofactors for their intrinsic fluorescence has meant that such proteins can be introduced into and expressed in cells via genetic constructs.
  • GFPs green fluorescent proteins
  • Miyawaki et al (1997) Nature 388, 882-887 describes a GFP-based Ca 2+ sensing system
  • Mitra et al (1996) Gene 173, 13-17 describes a rwo-GFP-based system for use in identifying protease inhibitors
  • WO 97/28261 discloses a two-GFP system in which the GFP donor and GFP acceptor are linked by a peptide containing a protease cleavage site.
  • WO 95/07463 describes uses of GFPs;
  • WO 96/23898 relates to a method of detecting biologically active substances using GFPs;
  • Heim & Tsien (1996) Current Biology 6, 178-182 relates to engineered GFPs with improved brightness, longer wavelengths and fluorescence resonance energy transfer (FRET);
  • Poppenborg et al (1997) J. Biotechnol. 58, 79-88 relates to GFPs as reporters for bioprocess monitoring; Park & Raines (1997) Protein Science 6, 2344-2349 relates to a GFP as a signal for protein-protein interactions; Niswender et al (1995) J.
  • Microscopy 180, 109-116 relates to quantitative imaging of GFP in cultured cells; Chalfie et al (1994) Science 263, 802-805 relates to GFP as a marker for gene expression; Hampton et al (1996) Proc. Natl. Acad. Sci.
  • USA 93, 828-833 relates to the in vivo examination of membrane protein localization and degradation with GFP; Heim et al (1995) Nature 373, 663-664 relates to mutant GFPs with altered fluorescent properties; Mosser et al (1997) BioTechniques 22, 150-161 relates to the use of a dicistronic expression cassette encoding GFP for the screening and selection of cells expressing inducible gene products; Suarez et al (1997) Gene 196, 69-74 relates to GFP-based reporter systems for genetic analysis of bacteria; Niedenthal et al (1996) Yeast 12, 773-778 relates to GFP as a marker for gene expression and subcellular localization in budding yeast; and Prescott et al (1997) FEBS Lett 411, 97-101 relates to the use of GFP as a marker for assembled mitochondrial ATP synthase in yeast.
  • variant GFPs which have improved properties, especially improved or new spectral properties, for use in biological systems, especially those where fluorescence resonance energy transfer (FRET) is used to study the biological system.
  • FRET fluorescence resonance energy transfer
  • a first aspect of the invention provides a polypeptide comprising the functional portion of green fluorescent protein but wherein valine (V) 163 has been replaced with alanine (A), serine (S) 175 has been replaced with glycine (G), isoleucine (I) 167 has been replaced with threonine (T), phenylalanine (F) 64 has been replaced with leucine (L), serine (S) 65 has been replaced with threonine (T), serine (S) 72 has been replaced with alanine (A), and threonine (T) 203 has been replaced with tyrosine (Y).
  • valine (V) 163 has been replaced with alanine (A)
  • serine (S) 175 has been replaced with glycine (G)
  • isoleucine (I) 167 has been replaced with threonine (T)
  • phenylalanine (F) 64 has been replaced with leucine (L)
  • serine (S) 65
  • functional portion we include the meaning that it is the portion of the protein which, in the absence of other portions of GFP, gives rise to useful fluorescent properties, such as the portion being fluorescent. It will be appreciated that, in respect of this first aspect of the invention the GFP or polypeptide comprising the functional portion of GFP with the given mutations may also include other mutations which may confer further desirable properties.
  • a second aspect of the invention provides a polypeptide which has the amino acid sequence
  • the difference with respect to the native GFP can be summarised as S2G, H25Q, S30R, F64L, S65T, S72A, Q80R, F84L, V163A, I167T, S175G, T203Y, K209Q, N212H, E213G, A216S, + 239S + 240F, making use of standard single letter amino acid code.
  • green fluorescent protein in the context of a single protein, we mean wild-type green fluorescent protein as described in Prasher et al (1992) Gene 111, 229-233 and whose amino acid sequence is given in Figure 1.
  • GFP may be used to denote variants which in fact fluoresce yellow or blue.
  • variant GFP The sequence of a particular preferred polypeptide of the invention (variant GFP) is shown in Figure 3, and its sequence is compared with A. victoria GFP and mm GFP5 (Zernicka-Goeta et al (1997) Development ⁇ 2A, 1133-1137 in Figures 4 and 5.
  • the functional portion of the polypeptide which contains the mutations as said may be incorporated into any suitable polypeptide in which it is desired to have a fluorescent moiety.
  • the functional portion is included in a polypeptide whose fluorescence or change in fluorescence is measured under suitable conditions.
  • the polypeptide may be one which is expressed as a reporter molecule (since its expression may be measured fluorimetrically).
  • the functional portion may be included in a polypeptide which is used in a biological system which makes use of FRET.
  • a polypeptide of the invention includes a polypeptide which contains the fluorescent portion as said, and it is used in conjunction with another fluorescent moiety with which it acts as a donor or acceptor in a FRET reaction.
  • the polypeptide of the invention contains, in addition to the functional portion as said, a further fluorescent moiety in the same polypeptide chain and the pair of fluorescent moieties may act as donor- acceptor pairs in a FRET reaction.
  • the polypeptide of the invention typically is a fusion protein containing at least the functional portion of the polypeptide which contains the mutations as said.
  • polypeptide of the invention may be used in any suitable prior art FRET method.
  • polypeptides of the invention are particularly suited for use in the FRET method described in UK Patent Application No 9817229.9 entitled “Fluorescent assay for biological systems" and the PCT application claiming priority from that application and which was filed on the same day as this application since the mutations confer an unusually high fluorescent lifetime.
  • the polypeptides of the invention are believed to be particularly suited as acceptor molecules since, at least in relation to the molecule of the second aspect of the invention, it excites at 514nm and emits at 531nm.
  • a particularly preferred polypeptide of the invention is one which has the amino acid sequence as shown in Figure 3.
  • a further preferred polypeptide of the invention is one which comprises at least residues 7 to 229 of green fluorescent protein containing said amino acid replacements.
  • the minimal domain required for fluorescence in GFP is believed to be amino acids 7 to 229 (Li et al (1997) J. Biol. Chem. 272, 28545-28549. Also, this information, and other information about GFPs, is available from Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303, USA (xqli@CLONTECH . com) .
  • a polypeptide which comprises at least residues 7 to 229 of green fluorescent protein not only contains the amino acid replacements described with respect to the first aspect of the invention, but also, compared to native A. victoria GFP, the amino acid replacements H25Q, S30R, Q80R, F84L, K209Q, N212H, F213G and A216S.
  • a still further preferred polypeptide is one comprising a further fluorescent moiety.
  • the further fluorescent moiety is one which is capable of FRET with the said portion of the variant GFP.
  • this further fluorescent moiety is a GFP or a variant GFP.
  • a particularly preferred polypeptide of the invention is a fusion polypeptide which contains at least the functional portion of the polypeptide which contains the mutations as said, and contains a further mutant GFP.
  • a third aspect of the invention provides a polynucleotide encoding a polypeptide of the first or second aspect of the invention.
  • the polynucleotide may be DNA or RNA; DNA is preferred.
  • a particularly preferred polynucleotide of the invention is shown in Figure 3 (DNA sequence) but, because of the degeneracy of the genetic code, it will be appreciated that other polynucleotides may encode the same polypeptide (ie with the amino acid sequence given in Figure 3).
  • a fourth aspect of the invention provides an expression vector encoding a polypeptide of the first or second aspect of the invention.
  • the expression vectors of the invention, and other polynucleotides can be constructed by standard laboratory molecular biology methods such as those described in Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York incorporated herein by reference.
  • the polynucleotide of the invention (typically DNA) may be expressed in a suitable host to produce a polypeptide comprising the polypeptide of the invention.
  • the DNA encoding the polypeptide of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention.
  • Such techniques include those disclosed in US Patent Nos.
  • polynucleotide such as DNA
  • encoding the polypeptide of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • bacteria for example E. coli and Bacillus subtilis
  • yeasts for example Saccharomyces cerevisiae
  • filamentous fungi for example Aspergillus
  • plant cells animal cells and insect cells.
  • the vectors include a prokaryotic replicon, such as the CoIEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types.
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p7rc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long te ⁇ ninal repeat to drive expression of the cloned gene.
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • a variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules. Synthetic linkers containing one _or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3' -single-stranded termini with their 3'-5'- exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.
  • the combination of these activities therefore generates blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying polymeric linker sequences at their ends.
  • These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.
  • a desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
  • the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • the present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).
  • Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650.
  • Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful.
  • reagents useful in transfecting such cells for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.
  • Electroporation is also useful for transforming cells and is well known in the art for traiiisfonning yeast cells, bacterial cells and vertebrate cells.
  • bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25 ⁇ FD.
  • Successfully transformed cells ie cells that contain a DNA construct of the present invention
  • cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention.
  • Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208.
  • the presence of the protein in the supernatant can be detected using antibodies.
  • Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
  • transformation and expression is indicated by the production of a fluorescent protein in this case.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • a particularly suitable "starting" vector is the pcDNA3.1 vector distributed by Invitrogen (Invitrogen BN, De Schelp 12, 9351 ⁇ N Leek,
  • Cytomegalovirus enhancer-promoter for high level expression of the insert in mammalian cells (the insert is for example the cDNA encoding the polypeptides described above and- has to be cloned into the vector); (ii) multiple cloning site in forward and reverse orientation; (iii) expression cassette for a selectable marker in eukaryotic cells (neomycin, zeocin or hygromycin).
  • polypeptide of the invention When the polypeptide of the invention is to be used ex vivo, such as in an in vitro assay or the like, it may be convenient to express the polypeptide in a bacterial system (such as E. coli), or in yeast or insect cells, or in other systems which have been designed for facile production of large amounts of protein.
  • a bacterial system such as E. coli
  • yeast or insect cells When the polypeptide of the invention is to be used in an in vivo assay it is conveniently expressed in the cell in which the assay is to be carried out, typically a mammalian cell.
  • DNA encoding the polypeptide of the invention may be fused to the promoter/enhancer elements of a gene under investigation.
  • DNA stably introduced into mammalian cells may be used as a reporter for expression of the respective gene under investigation.
  • Readout of the activity is the amount of polypeptide, such as YFP5, expressed, which can be determined by determination of the specific fluorescence of the polypeptide.
  • Similar DNAs may be generated for the other GFPs such as those listed in Table 1 (see below). Since they have overlapping spectra they cannot be used simultaneously in the same cells.
  • fluorescent lifetime imaging CFP, MmGFP5 and YFP5 for example, could be used simultaneously as their lifetimes are sufficiently separated from each other.
  • FLIM fluorescent lifetime imaging
  • Multifrequency FLIM is described in UK Patent Application No 9817227.3 entitled “Multiple Frequency Fluorescence Lifetime Imaging” and the PCT application claiming priority from that application and which has the same filing date as this application.
  • Figure 1 shows the cDNA and amino acid sequence of A. victoria green fluorescent protein (GFP).
  • Figure 2 shows the cDNA and amino acid sequence of a prior art mutant GFP (mmGFP5; Zernicka-Goetz et at).
  • Figure 3 shows the cDNA and amino acid sequence of a polypeptide of the invention (called mmYFP or mYFP5 or YFP5) which is described in more detail in Example 1.
  • Figure 4 is a comparison of the cDNA sequences from Figures 1 to 3.
  • Figure 5 is a comparison of the amino acid sequences from Figures 1 to 3.
  • Example 1 Construction of mutant GFP and its properties
  • MmGFP5 is a wtGFP mutated in V163A, S175G, I167T, F64L and S65T; the mutations N163A, S175G and I167T were introduced into wtGFP by Siemering et al (1996) Current Biol. 6, 1653-, and Zernicka- Goetz et al introduced the mutations F64L and S65T).
  • This approach introduced mutations S72A and T203Y into MmGFP5 using primer pairs
  • the final PCR product was gel-purified, digested with ⁇ coRI and Xb ⁇ l and subcloned into pEFT7MCS.
  • This vector is based on pEF-BOS (Nucleic Acids Res. (1990) Sep 11; 18(17), 5322 pEF-BOS, a powerful mammalian expression vector. Mizushima S, Nagata S).
  • pEF-BOS Nucleic Acids Res. (1990) Sep 11; 18(17), 5322 pEF-BOS, a powerful mammalian expression vector. Mizushima S, Nagata S).
  • a modified version of pEF-BOS containing a Neo resistance expression cassette, pEFl-Neo was obtained from G. Baier, Innsbruck.
  • the Neo expression cassette to make the vector smaller and introduced a T7 RNA polymerase promoter as well as several unique restriction enzyme sites downstream of the human EFl ⁇ promoter and the SV40 polyadenylation
  • Any other suitable vector, as described in the specification, may be used for the expression of the mutant GFP.
  • the introduced mutations were verified by sequencing using Sequenase.
  • the sequence of YFP5 is given in Figure 3.
  • the respective GFP mutants were expressed in cells by microinjection (Pepperkok et al, 1997 in “Microinjection and Transgenesis", eds. Cid- Arregui and Garcia-Carranca, Springer, Heidelberg, pp 145-154) of plasmids based on the vector pEFT7MCS and with inserts of the respective GFP encoding cDNAs.
  • microinjection Pieris et al, 1997 in "Microinjection and Transgenesis", eds. Cid- Arregui and Garcia-Carranca, Springer, Heidelberg, pp 145-154
  • plasmids based on the vector pEFT7MCS and with inserts of the respective GFP encoding cDNAs.
  • At 2h after microinjection cells were mounted on the FLIM microscope set-up and the respective lifetimes were determined at 37 °C in living cells. Any suitable expression system or lifetime-detection system may be used.
  • YFP5 shows a well-separated and significantly longer lifetime than other GFP mutants making it an ideal partner in multi-labelling FLIM experiments.
  • Table 1 Fluorescent lifetimes of various GFP mutants.

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Abstract

La présente invention concerne un polypeptide contenant la partie fonctionnelle d'une protéine à fluorescence verte mais dans laquelle la valine (V) 163 a été remplacée par l'alanine (A), la sérine (S) 175 a été remplacée par la glycine (G), l'isoleucine (I) 167 a été remplacée par la thréonine (T), la phénylalanine (F) 64 a été remplacée par la leucine (L), la sérine (S) 65 a été remplacée par la thréonine (T), la sérine (S) 72 a été remplacée par l'alanine (A), et la thréonine (T) 203 a été remplacée par la tyrosine (Y). La présente invention concerne aussi des polynucléotides qui codent pour cette protéine et des utilisations de la protéine comme marqueur.
EP99940293A 1998-08-08 1999-08-06 Proteine a fluorescence verte modifiee Withdrawn EP1102791A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
GB9817225 1998-08-08
GB9817227 1998-08-08
GBGB9817229.9A GB9817229D0 (en) 1998-08-08 1998-08-08 Fluorescent assay for biological systems
GBGB9817227.3A GB9817227D0 (en) 1998-08-08 1998-08-08 Multiple frequency fluorescence lifetime imaging
GB9817229 1998-08-08
GBGB9817225.7A GB9817225D0 (en) 1998-08-08 1998-08-08 Fluorescent protein
PCT/GB1999/002596 WO2000008054A1 (fr) 1998-08-08 1999-08-06 Proteine a fluorescence verte modifiee

Publications (1)

Publication Number Publication Date
EP1102791A1 true EP1102791A1 (fr) 2001-05-30

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EP99940295A Withdrawn EP1102977A1 (fr) 1998-08-08 1999-08-06 Mesures de fluorescence pour systemes biologiques
EP99940294A Withdrawn EP1102976A1 (fr) 1998-08-08 1999-08-06 Imagerie de duree de vie de fluorescence a des frequences multiples
EP99940293A Withdrawn EP1102791A1 (fr) 1998-08-08 1999-08-06 Proteine a fluorescence verte modifiee

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EP99940295A Withdrawn EP1102977A1 (fr) 1998-08-08 1999-08-06 Mesures de fluorescence pour systemes biologiques
EP99940294A Withdrawn EP1102976A1 (fr) 1998-08-08 1999-08-06 Imagerie de duree de vie de fluorescence a des frequences multiples

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JP4492826B2 (ja) 1996-11-20 2010-06-30 イントロジェン セラピューティクス，インコーポレイテッド アデノウイルスベクターの産生および精製のための改良された方法
CA2367400A1 (fr) 1999-04-06 2000-10-12 Yale University Analyse d'adresses fixes de sequences etiquetees
AU6770800A (en) 1999-08-13 2001-03-13 Yale University Analysis of sequence tags with hairpin primers
AU2001260625A1 (en) * 2000-08-14 2002-02-25 Michiyuki Matsuda Activity monitor protein for low-molecular weight gtp-binding protein
AU2001232219A1 (en) * 2000-08-14 2001-06-06 Michiyuki Matsuda Protein monitoring the activity of small gtp-binding protein
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WO2000008054A1 (fr) 2000-02-17
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EP1102977A1 (fr) 2001-05-30
WO2000008444A1 (fr) 2000-02-17
AU5429299A (en) 2000-02-28
AU5429199A (en) 2000-02-28
JP2002522040A (ja) 2002-07-23
WO2000008443A9 (fr) 2000-05-25
WO2000008443A1 (fr) 2000-02-17
EP1102976A1 (fr) 2001-05-30

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