WO2007140983A1 - PROTÉINES FLUORESCENTES wfCGFP ET LEUR UTILISATION - Google Patents

PROTÉINES FLUORESCENTES wfCGFP ET LEUR UTILISATION Download PDF

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WO2007140983A1
WO2007140983A1 PCT/EP2007/004967 EP2007004967W WO2007140983A1 WO 2007140983 A1 WO2007140983 A1 WO 2007140983A1 EP 2007004967 W EP2007004967 W EP 2007004967W WO 2007140983 A1 WO2007140983 A1 WO 2007140983A1
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protein
proteins
nucleic acid
amino acid
wfcgfp2
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English (en)
Inventor
Stefan Golz
Eugene Vysotski
Svetlana Markova
Ludmilla Burakova
Ludmilla Frank
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Bayer AG
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Bayer Healthcare AG
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
    • C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid

Definitions

  • the invention relates to the nucleotide and amino acid sequence, as well as the activity and use of the fluorescent proteins wfCGFPl, and wfCGFP2 (well-folded fluorescence protein of clytia gregaria 1 and 2).
  • a variety of coelenterates are bioluminescent (Morin et al., 1974) and emit blue or green light.
  • the aequorin from Aequoria victoria (Shimomura et al., 1962), identified as the first light-producing protein in 1962, emitted a blue light as an isolated protein and not the phenotypically observed green light of Aequoria victoria.
  • 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 hydrozoa (aequoria, halistaura obeiia) and anthropods (acanthotilum, sea cactus, cavernularia, renila, ptilosarcus, stylatula) (Morin et al., 1971; Morin et al., 1971 ⁇ , Wampler et al., 1971, Wampler et al., 1973, Cormier et al., 1973, 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.
  • FRET fluorescence resonance energy transfer
  • BRET Bioluminescence Resonance Energy Transfer
  • other energy transfer methods have previously been described in the literature (Mitra et al., 1996; Ward et al., 1978; Cardullo et al, 1988; US Patent No. 4,777,128; No. 5,126,508, U.S. Patent No. 4,927,923, U.S. Patent No. 5,279,943).
  • Further non-radioactive methods for energy transfer by means of GFP have also already been described (PCT appl. WO 98/02571 and WO 97/28261).
  • 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 genes 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
  • Luminescence refers to 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. Production of the mutants
  • the mutations were inserted using molecular biological methods.
  • the "quick change” method of the company Stratagene catalog number # 200521, revision # 06300 Ib, edition 2003 was used.
  • fluorescent proteins have already been described which have altered spectral, physicochemical or biochemical properties by replacing individual amino acids. These include EGFP (Falkow et al., 1996).
  • mutants wfCGFPl and wfCGFP2 When expressing the mutants wfCGFPl and wfCGFP2 in eukaryotic cells, it surprisingly turned out that stable fluorescent cell lines can be established with the aid of the mutants. This was previously not possible with a CFP.
  • the use of the mutants according to the invention wfCGFPl and wfCGFP2 consequently has the advantage that cells no longer need to be constantly transformed.
  • stable cell lines transformed with wfCGFPl and wfCGFP2 leads to lower variances in the test systems.
  • Another advantage is that a stable cell line can be easily transformed with other nucleic acids. Cells of a stable cell line are 100% transformed. Transient transformations have so far usually achieved transformation rates of 20-30%.
  • the fluorescent protein wfCGFPl has a combination of two mutations that lead to altered physicochemical and biochemical properties. These mutations are combinations of amino acid substitutions at position 103 with amino acid substitutions at other positions. At position 103, asparagine (N) was replaced by aspartic acid (D), hereinafter referred to as 103D. Surprisingly, the combination of 103D with amino acid substitutions at position 162 showed altered property. At position 162, a methionine (M) was replaced by isoleucine (I), hereinafter referred to as 1621. The combination of 103D and 1621 will be referred to as wfCGFPl.
  • the fluorescent protein wfCGFP2 has a combination of two mutations that lead to altered physicochemical and biochemical properties. These mutations are combinations of amino acid substitutions at position 103 Amino acid exchange at other positions. At position 103, asparagine (N) was replaced by aspartic acid (D), hereinafter referred to as 103D. Surprisingly, the combination of 103D with amino acid substitutions at position 163 showed altered properties. At position 163, a lysine (K) was replaced by an arginine (R), hereinafter referred to as 163R. The combination of 103D and 163R is referred to hereafter as wfCGFP2.
  • the fluorescent proteins wfCGFPl and wfCGFP2 show the highest homology at the amino acid level to CGFP from Clytia gregaria with an identity of 99%.
  • the fluorescent protein CGFP is described in WO2004052926 A1.
  • the fluorescent proteins wfCGFPl and wfCGFP2 show an altered expressibility in eukaryotic systems.
  • the invention relates to the fluorescent proteins wfCGFPl having the amino acid sequence represented by SEQ ID NO: 2.
  • the invention also relates to the nucleic acid molecule shown in SEQ ID NO: 1.
  • the invention relates to the fluorescent proteins wfCGFP2 having the amino acid sequence represented by SEQ DD NO: 6.
  • the invention also relates to the nucleic acid molecule shown in SEQ ID NO: 5.
  • the invention also relates to functional equivalents of the fluorescent protein wfCGFPl.
  • Functional equivalents are those proteins that have comparable physicochemical or biochemical properties.
  • the invention also relates to functional equivalents of the fluorescent protein wfCGFP2.
  • Functional equivalents are those proteins that have comparable physicochemical or biochemical properties.
  • the invention also relates to combinations of the amino acid substitution at position 103 with other amino acid substitutions.
  • the invention also relates to combinations of the amino acid substitution at position 103, other than aspartic acid, with other amino acid substitutions.
  • the invention also relates to combinations of amino acid replacement at position 162 with other amino acid substitutions.
  • the invention also relates to combinations of amino acid replacement at position 162, other than isoleucine, with other amino acid substitutions.
  • the invention also relates to combinations of amino acid replacement at position 163, with other amino acid substitutions.
  • the invention also relates to combinations of amino acid substitution at position 163, other than arginine, with other amino acid substitutions.
  • the invention also relates to the individual exchanges at position 103 as well as 103D deviating exchanges at position 103.
  • the invention also relates to the individual exchanges at position 162, as well as 1621 deviating exchanges at position 162.
  • the invention also relates to the individual exchanges at position 163, as well as 163R deviating exchanges at position 163.
  • the invention relates to CGFP proteins which, in the region of amino acid positions 93 to 112, preferably have 98 to 108, in particular 101 and 105, 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 EO NO: 3.
  • the invention relates to CGFP proteins which have in the range of amino acid positions 93 to 112 preferably 98 to 108, in particular 101 and 105 one or more amino acid substitutions and 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 CGFP proteins which have in the range of amino acid positions 93 to 112 preferably 98 to 108, in particular 101 and 105, one or more amino acid substitutions.
  • the invention also relates to chimeric proteins consisting of fragments of CGFP proteins which, in the region of the amino acid position 93 to 112, preferably 98 to 108, in particular 101 and 105 have one or more amino acid exchanges and other fluorescent proteins or fragments of other fluorescent proteins.
  • the invention relates to CGFP proteins which, in the region of amino acid positions 152 to 172, preferably 157 to 167, in particular 160 and 164, have 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 E) NO: 3.
  • the invention relates to CGFP proteins having in the region of amino acid positions 152 to 172 preferably 157 to 167, in particular 160 and 164 one or more amino acid substitutions and 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 CGFP proteins which have in the region of the amino acid positions 152 to 172 preferably 157 to 167, in particular 160 and 164, one or more amino acid substitutions.
  • the invention also relates to chimeric proteins consisting of fragments of CGFP proteins which in the region of amino acid position 152 to 172 preferably 157 to 167, in particular 160 and 164 have one or more amino acid substitutions and other fluorescent proteins or fragments of other fluorescent proteins.
  • the invention relates to CGFP proteins which, in the region of amino acid positions 153 to 173, preferably 158 to 168, in particular 161 and 165, have 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 have in the region of amino acid positions 153 to 173 preferably 158 to 168, in particular 161 and 165, one or more amino acid substitutions and 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 CGFP proteins which, in the region of amino acid positions 153 to 173, preferably 158 to 168, in particular 161 and 165, have one or more amino acid exchanges.
  • the invention also relates to chimeric proteins consisting of fragments of CGFP proteins which, in the region of the amino acid position 153 to 173, preferably 158 to 168, in particular 161 and 165, have 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. Also functional fragments of the fluorescent protein wfCGFPl protein or for such encoding nucleic acids according to the invention.
  • mutants of the fluorescent protein wfCGFPl protein or for such encoding nucleic acids according to the invention are also mutants of the fluorescent protein wfCGFPl protein or for such encoding nucleic acids according to the invention.
  • mutants of the fluorescent protein wfCGFP2 protein or for such encoding nucleic acids according to the invention are also possible.
  • the change of fluorescent proteins in the similar region of the protein structure are according to the invention.
  • mutant molecules wherein the altered physicochemical and biochemical properties result in cells being stably transfected with a nucleic acid construct containing a nucleic acid encoding such a mutant.
  • HCS high content screening
  • the proteins wfCGFPl and wfCGFP2 are suitable as reporter genes for cellular systems especially for receptors, for ion channels, for transporters, for transcription factors or for inducible systems.
  • the proteins wfCGFPl and wfCGFP2 are suitable as reporter genes in bacterial and eukaryotic systems, especially in mammalian cells, in bacteria, in yeasts, in bakulo, in plants
  • the proteins wfCGFPl and wfCGFP2 are suitable as reporter genes for cellular systems in combination with bioluminescent or chemiluminescent systems especially systems with luciferases, with oxygenases, with phosphatases.
  • the proteins wfCGFPl and wfCGFP2 are suitable as marker proteins, especially in the FACS (fluorescence activated cell sorter) sorting.
  • the proteins wfCGFPl and wfCGFP2 are suitable as fusion proteins especially for receptors, ion channels, transporters, transcription factors, proteinases, kinases, phosphodiesterases, hydrolases, peptidases, transferases, membrane proteins, glycoproteins.
  • the proteins wfCGFPl and wfCGFP2 are suitable for immobilization, in particular by antibodies, by biotin, by magnetic or magnetizable carriers.
  • the proteins wfCGFPl and wfCGFP2 are suitable as proteins for energy transfer systems, in particular 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 proteins wfCGFPl and wfCGFP2 are suitable for labeling substrates or ligands specifically for proteases, for kinases, for transferases, for transporters, for ion channels.
  • the proteins wfCGFPl and wfCGFP2 are suitable for expression in bacterial systems especially for titer determination, as substrates for biochemical systems especially for proteinases and kinases.
  • proteins wfCGFPl and wfCGFP2 are suitable as markers specifically coupled to antibodies coupled to enzymes coupled to receptors coupled to ion channels and other proteins.
  • the proteins wfCGFPl and wfCGFP2 are suitable as reporter genes in the pharmacological search for active substances, especially in HTS (High Throughput Screening).
  • the proteins wfCGFPl and wfCGFP2 are suitable as components of detection systems especially for ELISA (enzyme-linked immunosorbent assay), for immunohistochemistry, for Western blot, for confocal microscopy.
  • proteins wfCGFPl and wfCGFP2 are suitable as markers for the analysis of interactions especially 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 proteins wfCGFPl and wfCGFP2 are useful as markers or fusion proteins for expression in transgenic organisms, especially in mice, in rats, in hamsters and other mammals, in primates, in fish, in worms, in plants.
  • the proteins wfCGFPl and wfCGFP2 are suitable as markers or fusion proteins for the analysis of embryonic development.
  • the proteins wfCGFPl and wfCGFP2 are useful as markers via a coupling agent specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
  • the proteins wfCGFPl and wfCGFP2 are suitable as reporters coupled to nucleic acids, especially to DNA, to RNA.
  • the proteins wfCGFPl and wfCGFP2 are suitable as reporters coupled to proteins or peptides.
  • the wfCGFPl and wfCGFP2 proteins coupled to nucleic acids or peptides are particularly suitable as probes for Northern blots, for Southern blots, for Western blots, for ELISA, for nucleic acid sequencing, for protein analyzes, chip analyzes.
  • the proteins wfCGFPl and wfCGFP2 are suitable as markers of pharmacological formulations especially of infectious agents, of antibodies, of "small molecules".
  • the proteins wfCGFPl and wfCGFP2 are suitable for geological investigations especially for ocean, groundwater and river currents.
  • the proteins wfCGFPl and wfCGFP2 are suitable for expression in expression systems, especially in in vitro translation systems, in bacterial systems, in yeast systems, in Bakulo systems, in viral systems, in eukaryotic systems.
  • the proteins wfCGFPl and wfCGFP2 are suitable for the visualization of tissues or cells in surgical interventions, especially in invasive, non-invasive, minimally invasive.
  • the proteins wfCGFPl and wfCGFP2 are also suitable for the marking of tumor tissues and other phenotypically altered tissues, especially in the histological examination, during surgical procedures.
  • the invention also relates to the purification of the proteins wfCGFPl and wfCGFP2 specifically as a wild-type protein, as a fusion protein, as a mutagenized protein.
  • the invention also relates to the use of wfCGFP1 and wfCGFP2 in the field of cosmetics, especially bath preparations, lotions, soaps, body colors, toothpaste, body powders.
  • the invention also relates to the use of wfCGFPl and wfCGFP2 for coloring foodstuffs, bath additives, ink, textiles, plastics in particular.
  • the invention also relates to the use of wfCGFPl and wfCGFP2 for coloring paper, especially greetings cards, paper products, wallpaper, craft items.
  • the invention also relates to the use of wfCGFPl and wfCGFP2 for dyeing liquids especially for water pistols, for fountains, for drinks, for ice cream.
  • the invention also relates to the use of wfCGFPl and wfCGFP2 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 wfCGFPl and wfCGFP2.
  • 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 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 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 the fluorescent proteins wfCGFPl and wfCGFP2 having the amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 6 and the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 5.
  • 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 inventive fluorescent proteins.
  • 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 diagnosis.
  • 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 expressing 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.
  • a fluorescent protein characterized in that its sequence comprises the sequence shown in SEQ ID NO: 2 and SEQ ID NO: 6, as well as functional fragments thereof.
  • a nucleic acid molecule which encodes a protein is that its sequence comprises the sequence shown in SEQ ID NO: 2 and SEQ ID NO: 6, as well as functional fragments thereof.
  • a fluorescent protein according to the invention is characterized in that it comprises an amino acid sequence which is represented by SEQ ID NO: 4 but has one or more mutations in the region of positions 154 to 179 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.
  • a component of the invention is a method for expressing a polypeptide according to 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.
  • Also part of the invention is the use of a protein according to the invention as marker or reporter gene also in combination with one or more other markers or reporter gene proteins.
  • Also according to the invention is a fluorescent protein, characterized in that it has a faster folding time by insertion of one or more mutations compared to the original sequence.
  • 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.
  • the invention also relates to mutants of the fluorescent protein CGFP, which lead to the altered expression of the protein.
  • the invention also relates to mutants of the fluorescent protein CGFP, as well as to CGFP and wfCGFP, which allow for the establishment of stable cell lines expressing a fluorescent protein.
  • the mutant fluorescent protein wfCGFPl is encoded by the following nucleotide sequence (SEQ ID NO: 1):
  • the fluorescent protein CGFP is encoded by the following nucleotide sequence (SEQ E) NO: 3):
  • the fluorescent protein wfCGFP2 is encoded by the following nucleotide sequence (SEQ ID NO: 5):
  • FIG. 1 A first figure.
  • FIG. 1 shows the plasmid map of the vector pcDNA3-wfCGFPl.
  • FIG. 2 shows the plasmid map of the vector pcDNA3-wfCGFP2.
  • FIG. 3 shows the plasmid map of the vector pcDNA3-CGFP.
  • FIG. 4 shows the alignment of CGFP, wfCGFPl and wfCGFP2 at the amino acid level. (black: CGFP, blue: wfCGFP2, red: wfCGFPl). The positions 103, 162 and 163 are outlined in black.
  • Figure 5 shows the result of a FACS analysis of wfCGFP2. Left: Measuring points before
  • Sorting Right: Measuring points after sorting. Fluorescent measurement events in red.
  • Figure 6 shows a photograph of a micrograph of stable cells of a wfCGFP2 expressing CHO cell line.
  • Table 1 shows an overview of some fluorescent proteins. Given is the name, the organism from which the protein has been isolated and the identification number (Acc No.) of the database entry.
  • Table 2 shows an overview of some fluorescent proteins. Given is the organism from which the protein has been isolated, the excitation and emission wavelengths that have been determined in spectral analyzes.
  • Table 3 shows an overview of some fluorescent proteins. Given is the organism from which the protein has been isolated, the name of the fluorescent protein and a selection of patents or applications. Examples
  • the plasmid pQE7 from Qiagen was used as a vector for the preparation of the construct shown below.
  • the cloning was carried out using standard molecular biological methods.
  • the derivatives of the vector were named pQE7-wfCGFPl and pQE7-wfCGFP2.
  • the vectors pQE7-wfCGFPl and pQE7-wfCGFP2 were used to express wfCGFP in bacterial systems.
  • the plasmid pcDNA3.1 (+) from Clontech was used as a vector for the preparation of the construct shown below.
  • the cloning was carried out using standard molecular biological methods.
  • the derivative of the vector was designated as pcDNA3-wfCGFPl and pcDNA3-wfCGFP2.
  • the vectors pcDNA3-wfCGFPl and pcDNA3-wfCGFP2 were used to express wfCGFP in eukaryotic systems.
  • Fig. 1 shows the plasmid map of the vector pcDNA3-wfCGFPl.
  • Fig. 2 shows the plasmid map of the vector pcDNA3-wfCGFP2.
  • Fig. 3 shows the plasmid map of the vector pcDNA3-CGFP.
  • the bacterial expression was carried out in E. coli strain BL21 (DE3) by transformation of the bacteria with the expression plasmids pQE7-wfCGFPl and pQE7-wfCGFP2.
  • the transformed bacteria were incubated in LB medium at 37 ° C for 3 hours and the
  • the constitutive eukaryotic expression was carried out in CHO cells by transfecting the cells with the expression plasmids pcDNA3-wfCGFP1, pcDNA3-wfCGFP2, pcDNA3-CGFP and pcDNA3.1 (+) in transient experiments.
  • 10,000 cells per well in DMEM-F 12 medium were plated on 96-well microtiter plates and incubated overnight at 37 ° C.
  • the transfection was carried out using the Fugene 6 kit (Roche) according to the 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.
  • the transfected cells were selected with 2 mg / ml geneticin and the fluorescence of the clones was determined.
  • the constitutive eukaryotic expression was carried out in CHO cells by transfecting the cells with the expression plasmids pcDNA3-wf CGFP1, pcDNA3-wfCGFP2, pcDNA3-CGFP and pcDNA3.1 (+).
  • CHO cells were transfected using Fugene 6 (ROCHE) according to the manufacturer's instructions and incubated in DMEM-F 12 medium overnight at 37 ° C. After 24 and 48 hours, respectively, the cells were detached by Acutase (Invitrogene, carried out according to the manufacturer's instructions), pelleted by centrifugation and subsequently taken up in PBS + 0.5% BSA.
  • the FACS sorting was carried out with the device FACSCalibur (Becton Dickinson) according to the manufacturer. A total of 9xlOE7 cells were sorted. The eluted cells were pelleted by centrifugation, taken up in PBS and transferred to cell culture flasks. To produce stable cell lines, the sorted cells were selected with 2 mg / ml geneticin and the fluorescence of the clones was determined.
  • FIG. 5 shows the result of the FACS analysis in a graphical representation.
  • FIG. 4 shows the alignment of the fluorescent proteins CGFP, wfCGFPl and wfCGFP2 at the amino acid level.

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Abstract

L'invention concerne la séquence de nucléotides et d'acides aminés ainsi que l'activité et l'utilisation de mutants des protéines fluorescentes wfCGFP.
PCT/EP2007/004967 2006-06-07 2007-06-05 PROTÉINES FLUORESCENTES wfCGFP ET LEUR UTILISATION Ceased WO2007140983A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006026586.6 2006-06-07
DE200610026586 DE102006026586A1 (de) 2006-06-07 2006-06-07 Fluoreszierende Proteine wfCGFP, sowie deren Verwendung

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WO2007140983A1 true WO2007140983A1 (fr) 2007-12-13

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Citations (3)

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WO2000008054A1 (fr) * 1998-08-08 2000-02-17 Imperial Cancer Research Technology Limited Proteine a fluorescence verte modifiee
WO2004052926A1 (fr) * 2002-12-09 2004-06-24 Bayer Healthcare Ag Protéine fluorescente de clytia gregaria (cgfp) isolée et son utilisation
WO2006081976A1 (fr) * 2005-02-05 2006-08-10 Bayer Healthcare Ag Mutants de la proteine fluorescente cgfp et leur utilisation

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2000008054A1 (fr) * 1998-08-08 2000-02-17 Imperial Cancer Research Technology Limited Proteine a fluorescence verte modifiee
WO2004052926A1 (fr) * 2002-12-09 2004-06-24 Bayer Healthcare Ag Protéine fluorescente de clytia gregaria (cgfp) isolée et son utilisation
WO2006081976A1 (fr) * 2005-02-05 2006-08-10 Bayer Healthcare Ag Mutants de la proteine fluorescente cgfp et leur utilisation

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Title
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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 *
WARD W W: "BIOCHEMICAL AND PHYSICAL PROPERTIES OF GREEN FLUORESCENT PROTEIN", GREEN FLUORESCENT PROTEIN, APPLICATIONS AND PROTOCOLS, 1998, pages 45 - 70, XP009028583 *

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TW200817428A (en) 2008-04-16

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