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WO2006081976A1 - Mutants de la proteine fluorescente cgfp et leur utilisation - Google Patents

Mutants de la proteine fluorescente cgfp et leur utilisation Download PDF

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Publication number
WO2006081976A1
WO2006081976A1 PCT/EP2006/000669 EP2006000669W WO2006081976A1 WO 2006081976 A1 WO2006081976 A1 WO 2006081976A1 EP 2006000669 W EP2006000669 W EP 2006000669W WO 2006081976 A1 WO2006081976 A1 WO 2006081976A1
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Prior art keywords
protein
cgfp
fluorescent
marker
nucleic acid
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.)
Ceased
Application number
PCT/EP2006/000669
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German (de)
English (en)
Inventor
Stefan Golz
Eugene Vysotski
Svetlana Markova
Ludmilla Burakova
Ludmilla Frank
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.)
Bayer AG
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Bayer Healthcare AG
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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Publication of WO2006081976A1 publication Critical patent/WO2006081976A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C07K14/43504—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

Definitions

  • the invention relates to the nucleotide and amino acid sequence, as well as the activity and use of mutants of the fluorescent protein CGFP (fluorescence protein of clytia gregaria).
  • CGFP fluorescent protein of clytia gregaria
  • a variety of coelenterates are bioluminescent (Morin et al., 1974) and emit blue or green light.
  • Aequoria victoria (Shimomura et al., 1962), aequorin identified as the first light-producing protein in 1962, emitted blue light as an isolated protein and did not emit Aequoria victoria like the phenotypically observed green light.
  • the green fluorescent protein (GFP) could be isolated from Aequoria victoria, which causes the medusa to appear phenotypically green due to the stimulation by the aequorin (Johnson et al, 1962, Hastings et al., 1969, Inouye et al, 1994).
  • Green fluorescent proteins could be isolated from different organisms. These include the Hydozoa (aequoria, halistaura obelia) and anthropods (acanthotilum, sea cactus, cavernularia, renila, ptilosarcus, stylatula) (Morin et al., 1971, Morin et al., 1971 TL, Wampler et al., 1971, Wampler et al., 1973, Cormier et al., 197.3, Cormier et al., 1974, Levine et al., 1982).
  • the fluorescent proteins differ not only in their nucleotide and amino acid sequence, but also in their biochemical and physical properties.
  • the spectral characteristics of the fluorescent proteins may differ both on the exitation and on the emission side.
  • An overview of the spectra of the fluorescence and the excitation wavelength can be found in Table 2.
  • Fluorescent proteins are already being used in a wide variety of fields.
  • Fluorescence Resonance Energy Transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET) and other energy transfer methods has already been described in the literature (Mitra et al., 1996, Ward et al., 1978, Cardullo et al,
  • Reporter or indicator genes are generally genes whose gene products can easily be detected by simple biochemical or histochemical methods. There are at least two types of reporter genes.
  • Resistance genes are genes whose expression confers on a cell resistance to antibiotics or other substances whose presence in the growth medium leads to cell death when the resistance gene is absent.
  • reporter gene The products of reporter genes are used in genetic engineering as fused or unfused indicators. Among the most common reporter genes is beta-galactosidase (Alam et al., 1990), alkaline phosphatase (Yang et al., 1997, Cullen et al., 1992), luciferases, and other photoproteins (Shinomura, 1985, Phillips GN, 1997; Snowdowne et al., 1984).
  • Luminescence is the emission of photons in the visible spectral range, this being done by excited emitter molecules. In contrast to fluorescence, the energy is not supplied from outside in the form of radiation of shorter wavelength.
  • Chemiluminescence is a chemical reaction that leads to an excited molecule that glows when the excited electrons return to their ground state. When this reaction is catalyzed by an enzyme, it is called bioluminescence.
  • the enzymes involved in the reaction are generally referred to as luciferases.
  • the mutations were inserted using molecular biological methods.
  • the "Quick change” method of the company Stratagene catalog number # 200521; Revision # 063001b, edition 2003 was used.
  • fluorescent proteins have already been described that changed by the exchange of individual amino acids? have spectral, physicochemical or biochemical properties. These include EGFP (Falkow et al., 1996).
  • the physicochemical changes of the mutant are based on a changed maturation time.
  • the term "maturation time” (short maturation time) refers to the period of complete differentiation or formation of the functional structure, whereby protein folding and the formation of primary, secondary, or tertiary structures play a decisive role.
  • B ei expression of the mutant in CGFP in eukaryotic cells was surprisingly found that the proportion of fluorescent cell clones significantly increased.
  • the fluorescent protein in CGFP has a combination of two mutations leading to altered physicochemical and biochemical properties. These mutations are amino acid substitutions at positions 164 and 169. At position 164, an isoleucine (I) was replaced by an alanine (A) and at position 169 an alanine (A) was replaced by a cysteine (C).
  • the fluorescent protein in CGFP shows the highest homology at the amino acid level to CGFP from Clytia gregaria with a 99% identity (shown in Figure 4).
  • the fluorescent protein in the CGFP shows an altered fluorescence intensity.
  • the fluorescent protein in the CGFP shows altered expressibility in eukaryotic systems.
  • the invention relates to the fluorescent protein in CGFP having the amino acid sequence represented by SEQ ID NO: 2.
  • the invention also relates to the nucleic acid molecule represented by SEQ ID NO: 1.
  • the invention also relates to functional equivalents of the fluorescent protein in the CGFP.
  • Functional equivalents are those proteins that have comparable physicochemical properties.
  • the invention also relates to combinations of the amino acid replacement at position 164 with an amino acid substitution at position 169 by a cysteine-divergent amino acid.
  • the invention also relates to combinations of the amino acid substitution at position 169 with an amino acid substitution at position 164 by an alanine-deviating amino acid.
  • the invention also relates to combinations of the amino acid substitution at position 164 with one or more other mutations.
  • the invention also relates to combinations of amino acid replacement at position 169 with one or more other mutations.
  • the invention relates to CGFP proteins which, in the region of amino acid positions 154 to 179, preferably have 165 to 170, in particular 164 and 169, one or more amino acid substitutions which lead to altered biochemical or physicochemical properties.
  • the amino acid sequence of the fluorescent protein .CGFP is represented by SEQ ID NO: 4.
  • the invention also relates to the nucleic acid molecule shown in SEQ ID NO: 3.
  • the invention relates to CGFP proteins which, in the region of amino acid positions 154 to 179, preferably have 165 to 170, in particular 164 and 169 one or more amino acid substitutions in combination with one or more mutations outside the preferred range which lead to altered physicochemical or biochemical properties ,
  • the invention also relates to fragments of CGPF 'proteins which have in the region of amino acid positions 154 to 179 preferably 165 to 170, in particular 164 and 169 one or more amino acid substitutions.
  • the invention also relates to chimeric proteins consisting of fragments of CGFP proteins which have in the region of 154 to 179 preferably 165 to 170, in particular 164 and 169 one or more amino acid exchanges and other fluorescent proteins or fragments of other fluorescent proteins,
  • regions with a similar Motif here are those sequences that have an identity of 80%, preferably 90% in this area.
  • mutants of the fluorescent protein in the CGFP protein or for such encoding nucleic acids are according to the invention.
  • the change of fluorescent proteins in the similar region of the proteme structure are according to the invention.
  • HCS high content screening
  • the protein in CGFP is useful as reporter gene for cellular systems especially for receptors, for ion channels, for transporters, for transcription factors or for inducible systems.
  • the protein in CGFP is useful as a reporter gene in bacterial and eukaryotic systems, especially in mammalian cells, in bacteria, in yeasts, in bakulo, in plants
  • the protein in CGFP is useful as a reporter gene for cellular systems in combination with bioluminescent or chemiluminescent systems, especially systems with luciferases, with oxygenases, with phosphatases.
  • the protein in the CGFP is suitable as a marker protein, especially in the FACS (fluorescence activated cell sorter) sorting.
  • the protein in CGFP is useful as a fusion protein especially for receptors, ion channels, transporters, transcription factors, proteinases, kinases, phosphodiesterases, hydrolases, peptidases, transferases, membrane proteins, glycoproteins.
  • the protein in the CGFP is suitable for immobilization specifically by antibodies, by biotin, by magnetic or magnetizable carriers.
  • the protein imCGFP suitable as a protein for energy transfer systems, especially FRET (Fluorescence Resonance Energy Transfer), BRET- (Bioluminescence Resonance Energy Transfer), FET (field effect transistors), FP (fluorescence polarization), HTRF (Homogeneous time- resolved fluorescence) systems.
  • FRET Fluorescence Resonance Energy Transfer
  • BRET- Bioluminescence Resonance Energy Transfer
  • FET field effect transistors
  • FP fluorescence polarization
  • HTRF Homogeneous time- resolved fluorescence
  • the protein in CGFP is useful as a marker of substrates or ligands specifically for proteases, for kinases, for transferases, for transporters, for ion channels.
  • the protein in the CGFP is suitable for expression in bacterial systems specifically for titer determination, as substrates for biochemical systems specifically for proteinases and kinases.
  • the protein in CGEP is useful as a marker specifically coupled to antibodies coupled to enzymes coupled to Si receptors coupled to ion channels and other proteins.
  • the protein in the CGFP is suitable as a reporter gene in the pharmacological drug discovery, especially in HTS (High Throughput Screening).
  • the protein in CGFP is suitable as a component of detection systems especially for enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, western blot, confocal microscopy.
  • ELISA enzyme-linked immunosorbent assay
  • the protein in CGFP is useful as a marker for the analysis of interactions specifically for protein-protein interactions, for DNA-protein interactions, for DNA-RNA interactions, for RNA-RNA interactions, for RNA-protein interactions (DNA: deoxyribonucleic acid; RNA: ribonucleic acid).
  • the protein in CGFP is useful as a marker or fusion protein for expression in transgenic organisms, especially in mice, in rats, in hamsters and other mammals, in primates, in fish, in worms, in plants.
  • the protein in CGFP is useful as a marker or fusion protein for analysis of embryonic development.
  • the protein in CGFP is useful as a marker via a coupling agent specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
  • the protein in CGFP is useful as a reporter coupled to nucleic acids, especially DNA, RNA.
  • the protein in CGFP is useful as a reporter coupled to proteins or peptides.
  • the protein coupled to nucleic acids or peptides in the CGFP is useful as a probe especially for Northern blots, for Southern blots, for Western blots, for ELISA, for nucleic acid sequencing, for protein analyzes, chip analyzes.
  • the protein in CGFP is useful as a marker of pharmacological formulations especially of infectious agents, antibodies, "small molecules”.
  • the protein in the CGFP is useful as a geological survey especially for marine, groundwater and river currents.
  • the protein in CGFP is useful for expression in expression systems, especially in in vitro translation systems, in bacterial systems, in yeast systems, in i & coco systems, in viral systems, in eukaryotic systems.
  • the protein in CGFP is useful as a visualization of tissues or cells during surgery, especially invasive, non-invasive, minimally-invasive.
  • the protein in CGFP is also useful for labeling tumor tissues and other phenotypically altered tissues, especially in histological examination, in surgical procedures.
  • the invention also relates to the purification of the protein in the CGFP specifically as a wild-type protein, as a fusion protein, as a mutagenized protein.
  • the invention also relates to the use of imCGFP in the field of cosmetics especially bath preparations, lotions, soaps, body colors, toothpaste, body powders.
  • the invention also relates to the use of imCGFP for dyeing foodstuffs, bath additives, ink, textiles, plastics.
  • the invention also relates to the use of imCGFP for coloring paper, especially greetings cards, paper products, wallpapers, craft articles.
  • the invention also relates to the use of imCGFP for dyeing liquids especially for water pistols, for fountains, for drinks, for ice cream.
  • the invention also relates to the use of imCGFP for the manufacture of toys, especially finger paint, make-up.
  • the invention relates to organisms containing a vector according to the invention.
  • the invention relates to organisms expressing a polypeptide of the invention.
  • the invention relates to organisms expressing a functional equivalent of imCGFP.
  • the invention relates to methods for expression of the fluorescent invention
  • Polypeptides in bacteria, eukaryotic cells or in in vitro expression systems Polypeptides in bacteria, eukaryotic cells or in in vitro expression systems.
  • the invention also relates to methods for purifying / isolating a polypeptide of the invention. - *
  • the invention relates to peptides having more than 5 consecutive amino acids which are recognized immunologically by antibodies against the fluorescent proteins of the invention.
  • the invention relates to the use of the fluorescent proteins according to the invention as marker and reporter genes, in particular for the pharmacological drug discovery and diagnostics.
  • the invention relates to the fluorescent protein in CGFP having the amino acid sequence represented by SEQ ID NO: 2 and the nucleotide sequence represented by SEQ ID NO: 1.
  • the invention relates to organisms containing a vector according to the invention.
  • the invention relates to organisms expressing a polypeptide of the invention
  • the invention relates to methods for expression of the fluorescent polypeptides according to the invention in bacteria, eukaryotic cells or in in vitro expression systems.
  • the invention also relates to methods for purification / isolation of a fluorescent polypeptide according to the invention.
  • the invention relates to peptides having more than 5 consecutive amino acids, which are recognized immunologically by antibodies against the fluorescent proteins according to the invention.
  • the invention relates to the use of the fluorescent proteins according to the invention as marker and reporter genes, in particular for the pharmacological drug discovery and diagnostics.
  • the invention relates to organisms containing a vector according to the invention.
  • the invention relates to organisms expressing a polypeptide of the invention
  • the invention relates to methods for expression of the fluorescent polypeptides according to the invention in bacteria, eukaryotic cells or in in vitro expression systems. .
  • the invention also relates to methods for purification / isolation of a fluorescent polypeptide according to the invention.
  • the invention relates to peptides having more than 5 consecutive amino acids, which are recognized immunologically by antibodies against the fluorescent proteins according to the invention. _ ⁇
  • the invention relates to the use of the fluorescent proteins according to the invention as
  • Marker and reporter genes in particular for pharmacological drug discovery and diagnostics.
  • a fluorescent protein characterized in that its sequence comprises the sequence shown in SEQ ID NO: 2, as well as functional fragments thereof.
  • nucleic acid molecule which encodes a protein is that its sequence comprises the sequence shown in SEQ ID NO: 2, as well as functional fragments thereof.
  • a component of the invention is a fluorescent protein, characterized in that it comprises an amino acid sequence which is represented by the SEQ ID NO: 4, but in Range of positions 154 to 179 has one or more mutations, which lead to a faster folding time of the protein, as well as its functional fragments.
  • nucleic acid molecule which encodes a protein as described in the preceding section.
  • nucleic acid molecule as described in the preceding 4 sections, characterized in that it contains a functional promoter 5 'to the coding sequence.
  • a component of the invention is a recombinant RNA or DNA vector which comprises a nucleic acid as described in the preceding section.
  • an organism containing a vector is as described in the preceding section.
  • Component of the invention is a method of expressing a polypeptide of the invention in bacteria, eukaryotic cells, or in in vitro translation systems.
  • the invention also provides a method for purifying a polypeptide as described in the preceding section.
  • a component of the invention is the use of a nucleic acid according to the invention or 6 as a marker or reporter gene also in combination with one or more other markers or reporter genes.
  • a component of the invention is likewise the use of a protein according to the invention as marker or reporter gene also in combination with one or more other markers or
  • Also according to the invention is a fluorescent protein, characterized in that it has a faster folding time compared to the original sequence by insertion of one or more mutations.
  • Also according to the invention is a fluorescent protein, characterized in that it by
  • Also part of the invention are processes for the preparation of a fluorescent protein, characterized in that it incorporates one or more mutations by insertion of one or more mutations has changed folding time compared to the original sequence, wherein one or more mutations are introduced by conventional mutagenesis methods.
  • the invention relates to the use of the fluorescent proteins according to the invention as a component of homo- or heterodiimers or multimers of fluorescent proteins which are linked to one another directly or by linkers.
  • a change in the RNA stability or RNA folding of the fluorescent protein CGFP or mutants of the fluorescent protein CGFP can also lead to altered biochemical or physicochemical properties of the protein.
  • the mutant fluorescent protein in the CGFP is encoded by the following nucleotide sequence
  • nucleotides encoding amino acid positions 164 and 169 are underlined.
  • the fluorescent protein CGFP is encoded by the following nucleotide sequence (SEQ ID NO: 3):
  • Fig. 1 shows the plasmid map of the vector pTriplEX2-imCGFP.
  • Fig. 2 shows the plasmid map of the vector pcDNA3-imCGFP.
  • Fig. 3 shows the plasmid map of the vector pcDNA3-CGFP.
  • Fig. 4 shows the spectrum of imCGFP. Excitation spectrum (-); Fluorescence (); X
  • Axis wavelength in nm
  • Y axis relative fluorescence intensity
  • the plasmid pTriplEx2 Clontech was used as a vector for the preparation of the construct shown below.
  • the cloning was carried out in the SfII position of the expression vector using standard molecular biological methods.
  • the derivative of the vector was termed pTriplEx2-imCGFP.
  • the vector pTriplEx2-imCGFP was used to express imCGFP in bacterial systems.
  • Fig. 1 shows the plasmid map of the vector pTriplEX2-imCGFP.
  • the plasmid pcDNA3.1 (+) from Clontech was used as a vector for the preparation of the construct shown below.
  • the cloning was carried out in the BamHI / NotI position of the expression vector using standard molecular biological methods.
  • the derivative of the vector was designated pcDNA3-imCGFP.
  • the vector pcDNA3-imCGFP was used to express imCGFP in eukaryotic systems.
  • Fig. 2 shows the plasmid map of the vector pcDNA3-inCGFP.
  • Bacterial expression was carried out in E. coli strain BL21 (DE3) by transformation of the bacteria with the expression plasmids pTriplEX2-imCGFP and pTriplEX2. The transformed bacteria were incubated in LB medium at 37 0 C for 3 hours and the expression for 4
  • the constitutive eukaryotic expression was carried out in CHO cells by transfecting the cells with the expression plasmids pcDNA3-imCGFP, pcDNA3-CGFP and pcDNA3.1 (+) in transient experiments.
  • 10,000 cells per well in DMEM-F12 medium were plated on 96-well microtiter plates and incubated overnight at 37 ° C.
  • the transfection took place with Help of the Fugene 6 kit (Roche) according to manufacturer's instructions.
  • the transfected cells were incubated overnight at 37 ° C in DMEM-F12 medium.
  • the fluorescence was measured in the fluorometer at room temperature.
  • Table 4 summarizes the results of the comparison. Shown are the number of fluorescent cells after transient transfection of CHO cells with the vectors pcDNA3-CGFP or pcDNA3-imCGFP or pcDNA3 (+) [without cDNA insertion].
  • the numbers given in the columns pcDNA3-CGFP, pcDNA3-imCGFP and pcDNA3 correspond to the number of fluorescent cells after transient transfection with the corresponding vector (of 10,000 transfected cells).
  • Table 5 summarizes the results of the study. Shown are the number of fluorescent bacterial clones at 20 0 C and 37 0 C.
  • the numbers given in the columns 2O 0 C and 37 0 C correspond to the number of fluorescent bacteriaHonen after transfection with the vectors pTriplEx2-CGFP or ⁇ TriplEx2- imCGFP and subsequent induction.
  • E. coli BL21 (DE3) were transformed with the plasmids pTriplEX2-CGFP or pTriplEX2-imCGFP.
  • the induction was carried out by the addition of - 1 mM IPTG and an incubation of 4 hours at 37 0 C.
  • the bacteria were harvested and resuspended in PBS.
  • the lysis was carried out by ultrasound. Subsequently, the fluorescence was measured in the fluorometer.
  • FIG. 4 shows the excitation and emission of the imCGFP

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Abstract

L'invention concerne la séquence nucléotidique et la séquence d'acides aminés, ainsi que l'activité et l'utilisation de mutants de la protéine fluorescente CGFP.
PCT/EP2006/000669 2005-02-05 2006-01-26 Mutants de la proteine fluorescente cgfp et leur utilisation Ceased WO2006081976A1 (fr)

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DE102005005438.2 2005-02-05
DE200510005438 DE102005005438A1 (de) 2005-02-05 2005-02-05 Mutanten des fluoreszierenden Proteins CGFPs, sowie deren Verwendung

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007140983A1 (fr) * 2006-06-07 2007-12-13 Bayer Healthcare Ag PROTÉINES FLUORESCENTES wfCGFP ET LEUR UTILISATION

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020078A1 (fr) * 1995-11-30 1997-06-05 Maxygen, Inc. Procede d'elaboration de polynucleotides presentant des caracteristiques desirees par selection iterative et recombinaison
WO2000008054A1 (fr) * 1998-08-08 2000-02-17 Imperial Cancer Research Technology Limited Proteine a fluorescence verte modifiee
WO2002077011A2 (fr) * 2001-03-12 2002-10-03 The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Variants proteiques vert fluorescent, sensibles a l'oxydoreduction
WO2004052926A1 (fr) * 2002-12-09 2004-06-24 Bayer Healthcare Ag Protéine fluorescente de clytia gregaria (cgfp) isolée et son utilisation

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
WO1997020078A1 (fr) * 1995-11-30 1997-06-05 Maxygen, Inc. Procede d'elaboration de polynucleotides presentant des caracteristiques desirees par selection iterative et recombinaison
WO2000008054A1 (fr) * 1998-08-08 2000-02-17 Imperial Cancer Research Technology Limited Proteine a fluorescence verte modifiee
WO2002077011A2 (fr) * 2001-03-12 2002-10-03 The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Variants proteiques vert fluorescent, sensibles a l'oxydoreduction
WO2004052926A1 (fr) * 2002-12-09 2004-06-24 Bayer Healthcare Ag Protéine fluorescente de clytia gregaria (cgfp) isolée et son utilisation

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Title
CORMACK B P ET AL: "FACS-OPTIMIZED MUTANTS OF THE GREEN FLUORESCENT PROTEIN (GFP)", GENE, ELSEVIER, AMSTERDAM, NL, vol. 173, 1996, pages 33 - 38, XP002919305, ISSN: 0378-1119 *
DELAGRAVE S ET AL: "RED-SHIFTED EXCITATION MUTANTS OF THE GREEN FLUORESCENT PROTEIN", BIO/TECHNOLOGY, NATURE PUBLISHING CO. NEW YORK, US, vol. 13, 1 February 1995 (1995-02-01), pages 151 - 154, XP000574465, ISSN: 0733-222X *
EHRIG T ET AL: "GREEN-FLUORESCENT PROTEIN MUTANTS WITH ALTERED FLUORENCE EXCITATION SPECTRA", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 367, 19 June 1995 (1995-06-19), pages 163 - 166, XP000579119, ISSN: 0014-5793 *
HEIM R ET AL: "ENGINEERING GREEN FLUORESCENT PROTEIN FOR IMPROVED BRIGHTNESS, LONGER WAVELENGTHS AND FLUROESCENCE RESONANCE ENERGY TRANSFER", CURRENT BIOLOGY, CURRENT SCIENCE,, GB, vol. 6, no. 2, 1 February 1996 (1996-02-01), pages 178 - 182, XP000676582, ISSN: 0960-9822 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007140983A1 (fr) * 2006-06-07 2007-12-13 Bayer Healthcare Ag PROTÉINES FLUORESCENTES wfCGFP ET LEUR UTILISATION

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TW200640945A (en) 2006-12-01

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