WO2007140983A1 - FLUORESCENT PROTEINS wfCGFP, and use thereof - Google Patents

FLUORESCENT PROTEINS wfCGFP, and use thereof Download PDF

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Publication number
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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Prior art keywords
protein
proteins
nucleic acid
amino acid
wfcgfp2
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PCT/EP2007/004967
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German (de)
French (fr)
Inventor
Stefan Golz
Eugene Vysotski
Svetlana Markova
Ludmilla Burakova
Ludmilla Frank
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Bayer Healthcare Ag
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Publication of WO2007140983A1 publication Critical patent/WO2007140983A1/en

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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.

Abstract

The invention relates to the nucleotide sequence and amino acid sequence, and to the activity and use of mutants of the fluorescent proteins wfCGFP.

Description

Fluoreszierende Proteine wfCGFP. sowie deren VerwendungFluorescent proteins wfCGFP. as well as their use
Die Erfindung betrifft die Nukleotid- und Aminosäuresequenz, sowie die Aktivität und Verwendung der fluoreszierenden Proteine wfCGFPl, sowie wfCGFP2 (well-folded fluorescence protein of clytia gregaria 1 und 2).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).
Fluoreszierende ProteineFluorescent proteins
Eine Vielzahl an Coelenteraten sind biolumineszent (Morin et al., 1974) und emittieren blaues oder grünes Licht. Das 1962 als erstes Licht produzierendes Protein identifizierte Aequorin aus Aequoria victoria (Shimomura et al., 1962) emittierte als isoliertes Protein ein blaues Licht und nicht das phenotypisch beobachtete grüne Licht von Aequoria victoria. Später konnte das grün fluoreszierende Protein (GFP) aus Aequoria victoria isoliert werden, das Aufgrund der Anregung durch das Aequorin die Meduse phenotypisch grün erscheinen lässt (Johnson et al, 1962; Hastings et al., 1969; Inouye et al, 1994).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. Later, 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).
Grün fluoreszierende Proteine konnten aus unterschiedlichen Organismen isoliert werden. Hierzu zählen die Hydrozoa (aequoria, halistaura obeiia) und Anthropoden (acanthotilum, sea cactus, cavernularia, renila, ptilosarcus, stylatula) (Morin et al., 1971; Morin et al., 1971 π, Wampler et al., 1971, Wampler et al., 1973, Cormier et al., 1973, Cormier et al., 1974, Levine et al., 1982).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 π, Wampler et al., 1971, Wampler et al., 1973, Cormier et al., 1973, Cormier et al., 1974, Levine et al., 1982).
Eine Zusammenfassung einiger fluoreszierender Proteine findet sich in Tabelle 1 :A summary of some fluorescent proteins can be found in Table 1:
Die fluoreszierenden Proteine unterscheiden sich nicht nur aufgrund ihrer Nukleotid- und Aminosäuresequenz, sondern auch aufgrund ihrer biochemischen und physikalischen Eigen- Schäften. Die spektralen Charakteristika der fluoreszierenden Proteine können sich sowohl auf der Exitations- als auch auf der Emmisionsseite unterscheiden. Eine Übersicht der Spektren der Fluoreszenz und der Anregungswellenlänge findet sich in Tabelle 2.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.
Die Verwendung von fluoreszierenden Proteinen wurde bereits zuvor beschrieben. Eine Übersicht findet sich in Tabelle 3 :The use of fluorescent proteins has been previously described. An overview can be found in Table 3:
Es konnte gezeigt werden, dass durch die Veränderung der Aminosäuresequenz von fluoreszierenden Proteinen die physikalischen und biochemischen Eigenschaften verändert werden können. Beispiele von mutagenisierten fluoreszierenden Proteinen sind in der Literatur beschrieben (Delagrave et al., 1995; Ehrig et al., 1995; Heim et al., 1996).It has been shown that by altering the amino acid sequence of fluorescent proteins, the physical and biochemical properties can be changed. Examples of mutagenized fluorescent proteins are described in the literature (Delagrave et al., 1995; Ehrig et al., 1995; Heim et al., 1996).
Fluoreszierende Proteine finden bereits in unterschiedlichsten Gebieten eine Anwendung. Die Verwendung von fluoreszierende Proteinen beim 'Fluorescence Resonance Energy Tranfer (FRET), Bioluminescence Resonance Energy Transfer (BRET) und anderen Energietransferverfahren wurde bereits in der Literatur beschrieben (Mitra et al., 1996; Ward et al., 1978; Cardullo et al, 1988; US patent no. 4,777,128; US patent no. 5,126,508; US patent no. 4,927,923; US patent no. 5,279,943). Weitere Nicht-radioaktive Methoden zum Energietransfer mittels GFP wurden in ebenfalls bereits beschrieben (PCT appl. WO 98/02571 and WO 97/28261).Fluorescent proteins are already being used in a wide variety of fields. The use of fluorescent proteins in fluorescence resonance energy transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET), and 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).
Reportersystemereporter systems
Als Reporter- oder Indikatorgene bezeichnet man generell Gene, deren Genprodukte sich mit Hilfe einfacher biochemischer oder histochemischer Methoden leicht nachweisen lassen. Man unterscheidet mindestens 2 Typen von Reportergenen.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.
1. Resistenzgene. Als Resistenzgene werden Gene bezeichnet, deren Expression einer Zelle die Resistenz gegen Antibiotika oder andere Substanzen verleiht, deren Anwesenheit im Wachstumsmedium zum Zelltod führt, wenn das Resistenzgen fehlt.1. resistance 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.
2. Reportergene. Die Produkte von Reportergenen werden in der Gentechnologie als fusionierte oder unfusionierte Indikatoren verwendet. Zu den gebräuchlichsten Reportergenen gehört die beta-Galaktosidase (Alam et al., 1990), alkalische Phosphatase2. 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
(Yang et al., 1997; Cullen et al., 1992), Luciferasen und andere Photoproteine (Shinomura,(Yang et al., 1997, Cullen et al., 1992), luciferases and other photoproteins (Shinomura,
1985; Phillips GN, 1997; Snowdowne et al., 1984)., 1985; Phillips GN, 1997; Snowdowne et al., 1984).
Als Lumineszenz bezeichnet man die Abstrahlung von Photonen im sichtbaren Spektralbereich, wobei diese durch angeregte Emittermoleküle erfolgt. Im Unterschied zur Fluoreszenz wird hierbei die Energie nicht von Außen in Form von Strahlung kürzerer Wellenlänge zugeführt.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.
Man unterscheidet Chemilumineszenz und Biolumineszenz. Als Chemolumineszenz bezeichnet man eine chemische Reaktion die zu einem angeregten Molekül führt, das selbst leuchtet, wenn die angeregten Elektronen in den Grundzustand zurückkehren. Wird diese Reaktion durch ein Enzym katalysiert, spricht man von Biolumineszenz. Die an der Reaktion beteiligten Enzyme werden generell als Luziferasen bezeichnet. Herstellung der MutantenA distinction is made between chemiluminescence and bioluminescence. 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
Zur Herstellung der Mutanten wurde mit Hilfe molekularbiologische Methoden die Mutationen eingefügt. Hierzu wurde das "Quick change" Verfahren der Firma Stratagene (Katalog Nummer #200521; Revision #06300 Ib; Auflage 2003) verwendet.To prepare the mutants, the mutations were inserted using molecular biological methods. For this purpose, the "quick change" method of the company Stratagene (catalog number # 200521, revision # 06300 Ib, edition 2003) was used.
wfCGFPwfCGFP
In der Literatur wurden bereits fluoreszierende Proteine beschrieben, die durch Austausch einzelner Aminosäuren veränderte spektrale, physikochemische oder biochemische Eigenschaften aufwiesen. Zu diesen gehört EGFP (Falkow et al., 1996).In the literature, 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).
Durch Einfügen von Mutationen in das zuvor beschriebene fluoreszente Protein CGFP konnte überrascherweise die Mutanten wfCGFPl sowie wfCGFP2 identifiziert werden, die veränderte Eigenschaften aufweisen.By inserting mutations into the previously described fluorescent protein CGFP, it was possible, surprisingly, to identify the mutants wfCGFPl and wfCGFP2 which have altered properties.
Bei der Expression der Mutanten wfCGFPl sowie wfCGFP2 in eukaryotischen Zellen stellte sich überraschenderweise heraus, das sich mit Hilfe der Mutanten stabile fluoreszierende Zelllinien etablieren lassen. Dies war bislang mit einem GFP nicht möglich. Die Verwendung der erfindungsgemäßen Mutanten wfCGFPl sowie wfCGFP2 hat folglich den Vorteil, dass Zellen nicht mehr ständig transformiert werden müssen. Des weiteren führt die Verwendung von stabilen Zelllinien, die mit wfCGFPl sowie wfCGFP2 transformiert sind, zu geringeren Varianzen in den Testsystemen. Ein weiterer Vorteil ist, das eine stabile Zelllinie leicht mit weiteren Nukleinsäuren transformiert werden kann. Zellen einer stabilen Zelllinie sind zu 100% transformiert. Durch transiente Transformationen konnten bislang üblicherweise Transformationsraten von 20 - 30% erreicht werden.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. Furthermore, the use of 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%.
Das fluoreszente Protein wfCGFPl weist eine Kombination von zwei Mutationen auf, die zu veränderten physikochemischen und biochemischen Eigenschaften führen. Bei diesen Mutationen handelt es sich um Kombinationen von Aminosäureaustauschen an der Position 103 mit Aminosäureaustauschen an anderen Positionen. An Position 103 wurde ein Asparagin (N) durch eine Asparaginsäure (D) ersetzt, im weiteren als 103D bezeichnet. Überraschenderweise zeigte die Kombination von 103D mit Aminosäureaustauschen an der Position 162 veränderte Eigenschaft. An Postion 162 wurde ein Methionin (M) durch ein Isoleucin (I) ersetzt, im weiteren als 1621 bezeichnet. Die Kombination aus 103D und 1621 wird im weiteren als wfCGFPl bezeichnet.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.
Das fluoreszente Protein wfCGFP2 weist eine Kombination von zwei Mutationen auf, die zu veränderten physikochemischen und biochemischen Eigenschaften führen. Bei diesen Mutationen handelt es sich um Kombinationen von Aminosäureaustauschen an der Position 103 mit Aminosäureaustauschen an anderen Positionen. An Position 103 wurde ein Asparagin (N) durch eine Asparaginsäure (D) ersetzt, im weiteren als 103D bezeichnet. Überraschenderweise zeigte die Kombination von 103D mit Aminosäureaustauschen an der Position 163 veränderte Eigenschaft. An Postion 163 wurde ein Lysin (K) durch ein Arginin (R) ersetzt, im weiteren als 163R bezeichnet. Die Kombination aus 103D und 163R wird im weiteren als wfCGFP2 bezeichnet.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.
Die fluoreszenten Proteine wfCGFPl sowie wfCGFP2 zeigen die höchste Homologie auf Aminosäureebene zu CGFP aus Clytia gregaria mit einer Identität von 99 %. Das fluoreszente Protein CGFP ist in WO2004052926 Al beschrieben.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.
Die fluoreszenten Proteine wfCGFPl sowie wfCGFP2 zeigen eine veränderte Expremierbarkeit in eukaryotischen Systemen.The fluorescent proteins wfCGFPl and wfCGFP2 show an altered expressibility in eukaryotic systems.
Die Erfindung betrifft die fluoreszenten Proteine wfCGFPl mit der Aminosäuresequenz repräsentiert durch SEQ ID NO: 2. Ebenfalls betrifft die Erfindung das Nukleinsäuremolekül dargestellt in SEQ ID NO: 1.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.
Die Erfindung betrifft die fluoreszenten Proteine wfCGFP2 mit der Aminosäuresequenz repräsentiert durch SEQ DD NO: 6. Ebenfalls betrifft die Erfindung das Nukleinsäuremolekül dargestellt in SEQ DD NO: 5.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.
Die Erfindung betrifft auch funktionelle Äquivalente des fluoreszenten Proteins wfCGFPl. Funktionelle Äquivalente sind solche Proteine, die vergleichbare physikochemische oder biochemische Eigenschaften aufweisen.The invention also relates to functional equivalents of the fluorescent protein wfCGFPl. Functional equivalents are those proteins that have comparable physicochemical or biochemical properties.
Die Erfindung betrifft auch funktionelle Äquivalente des fluoreszenten Proteins wfCGFP2. Funktionelle Äquivalente sind solche Proteine, die vergleichbare physikochemische oder biochemische Eigenschaften aufweisen.The invention also relates to functional equivalents of the fluorescent protein wfCGFP2. Functional equivalents are those proteins that have comparable physicochemical or biochemical properties.
Die Erfindung betrifft auch Kombinationen aus dem Aminosäureaustausch an Position 103 mit anderen Aminosäureaustauschen.The invention also relates to combinations of the amino acid substitution at position 103 with other amino acid substitutions.
Die Erfindung betrifft auch Kombinationen aus dem Aminosäureaustausch an Position 103, abweichend von Asparaginsäure, mit anderen Aminosäureaustauschen.The invention also relates to combinations of the amino acid substitution at position 103, other than aspartic acid, with other amino acid substitutions.
Die Erfindung betrifft auch Kombinationen aus dem Aminosäureaustausch an Position 162 mit anderen Aminosäureaustauschen.The invention also relates to combinations of amino acid replacement at position 162 with other amino acid substitutions.
Die Erfindung betrifft auch Kombinationen aus dem Aminosäureaustausch an Postion 162, abweichend von Isoleucin, mit anderen Aminosäureaustauschen. Die Erfindung betrifft auch Kombinationen aus dem Aminosäureaustausch an Postion 163, mit anderen Aminosäureaustauschen.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.
Die Erfindung betrifft auch Kombinationen aus dem Aminosäureaustausch an Postion 163, abweichend von Arginin, mit anderen Aminosäureaustauschen.The invention also relates to combinations of amino acid substitution at position 163, other than arginine, with other amino acid substitutions.
Die Erfindung betrifft auch die Einzelaustausche an Position 103, sowie von 103D abweichenden Austauschen an Position 103.The invention also relates to the individual exchanges at position 103 as well as 103D deviating exchanges at position 103.
Die Erfindung betrifft auch die Einzelaustausche an Position 162, sowie von 1621 abweichenden Austauschen an Position 162.The invention also relates to the individual exchanges at position 162, as well as 1621 deviating exchanges at position 162.
Die Erfindung betrifft auch die Einzelaustausche an Position 163, sowie von 163R abweichenden Austauschen an Position 163.The invention also relates to the individual exchanges at position 163, as well as 163R deviating exchanges at position 163.
Die Erfindung betrifft CGFP Proteine, die im Bereich der Aminosäurepositionen 93 bis 112 bevorzugt 98 bis 108, insbesondere 101 und 105 eine oder mehrere Aminosäureaustausche aufweisen, welche zu veränderten biochemischen oder physikochemischen Eigenschaften führen. Die Aminosäuresequenz des fluoreszenten Proteins CGFP ist repräsentiert durch SEQ ED NO: 4. Ebenfalls betrifft die Erfindung das Nukleinsäuremolekül dargestellt in SEQ EO NO: 3.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.
Die Erfindung betrifft CGFP Proteine, die im Bereich der Aminosäurepositionen 93 bis 112 bevorzugt 98 bis 108, insbesondere 101 und 105 eine oder mehrere Aminosäureaustausche aufweisen und in Kombination mit einer oder mehreren Mutationen außerhalb des bevorzugten Bereichs, die zu veränderten physikochemischen oder biochemischen Eigenschaften führen.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.
Die Erfindung betrifft auch Fragmente von CGFP Proteinen, die im Bereich der Aminosäurepositionen 93 bis 112 bevorzugt 98 bis 108, insbesondere 101 und 105, eine oder mehrere Aminosäureaustausche aufweisen.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.
Die Erfindung betrifft auch chimäre Proteine bestehend aus Fragmenten von CGFP Proteinen die im Bereich der Aminosäureposition 93 bis 112 bevorzugt 98 bis 108, insbesondere 101 und 105 eine oder mehrere Aminosäureaustausche aufweisen und anderen fluoreszenten Proteinen oder Fragmenten anderer fluoreszenter Proteine.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.
Die Erfindung betrifft CGFP Proteine, die im Bereich der Aminosäurepositionen 152 bis 172 bevorzugt 157 bis 167, insbesondere 160 und 164 eine oder mehrere Aminosäureaustausche aufweisen, welche zu veränderten biochemischen oder physikochemischen Eigenschaften führen. Die Aminosäuresequenz des fluoreszenten Proteins CGFP ist repräsentiert durch SEQ ID NO: 4. Ebenfalls betrifft die Erfindung das Nukleinsäuremolekül dargestellt in SEQ E) NO: 3.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.
Die Erfindung betrifft CGFP Proteine, die im Bereich der Aminosäurepositionen 152 bis 172 bevorzugt 157 bis 167, insbesondere 160 und 164 eine oder mehrere Aminosäureaustausche aufweisen und in Kombination mit einer oder mehreren Mutationen außerhalb des bevorzugten Bereichs, die zu veränderten physikochemischen oder biochemischen Eigenschaften führen.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.
Die Erfindung betrifft auch Fragmente von CGFP Proteinen, die im Bereich der Aminosäurepositionen 152 bis 172 bevorzugt 157 bis 167, insbesondere 160 und 164, eine oder mehrere Aminosäureaustausche aufweisen.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.
Die Erfindung betrifft auch chimäre Proteine bestehend aus Fragmenten von CGFP Proteinen die im Bereich der Aminosäureposition 152 bis 172 bevorzugt 157 bis 167, insbesondere 160 und 164 eine oder mehrere Aminosäureaustausche aufweisen und anderen fluoreszenten Proteinen oder Fragmenten anderer fluoreszenter Proteine.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.
Die Erfindung betrifft CGFP Proteine, die im Bereich der Aminosäurepositionen 153 bis 173 bevorzugt 158 bis 168, insbesondere 161 und 165 eine oder mehrere Aminosäureaustausche aufweisen, welche zu veränderten biochemischen oder physikochemischen Eigenschaften führen. Die Aminosäuresequenz des fluoreszenten Proteins CGFP ist repräsentiert durch SEQ ED NO: 4. Ebenfalls betrifft die Erfindung das Nukleinsäuremolekül dargestellt in SEQ DD NO: 3.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.
Die Erfindung betrifft CGFP Proteine, die im Bereich der Aminosäurepositionen 153 bis 173 bevorzugt 158 bis 168, insbesondere 161 und 165 eine oder mehrere Aminosäureaustausche aufweisen und in Kombination mit einer oder mehreren Mutationen außerhalb des bevorzugten Bereichs, die zu veränderten physikochemischen oder biochemischen Eigenschaften führen.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.
Die Erfindung betrifft auch Fragmente von CGFP Proteinen, die im Bereich der Aminosäurepositionen 153 bis 173 bevorzugt 158 bis 168, insbesondere 161 und 165, eine oder mehrere Aminosäureaustausche aufweisen.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.
Die Erfindung betrifft auch chimäre Proteine bestehend aus Fragmenten von CGFP Proteinen die im Bereich der Aminosäureposition 153 bis 173 bevorzugt 158 bis 168, insbesondere 161 und 165 eine oder mehrere Aminosäureaustausche aufweisen und anderen fluoreszenten Proteinen oder Fragmenten anderer fluoreszenter Proteine.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.
Als Bereiche mit ähnlichem Motif gelten hier solche Sequenzen, die in diesem Bereich eine Identität von 80%, bevorzugter Weise von 90% aufweisen. Ebenfalls sind funktionelle Fragmente des fluoreszenten Proteins wfCGFPl Proteins bzw. für solche kodierende Nukleinsäuren erfindungsgemäß.As 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.
Ebenfalls sind funktionelle Fragmente des fluoreszenten Proteins wfCGFP2 Proteins bzw. für solche kodierende Nukleinsäuren erfindungsgemäß.Also functional fragments of the fluorescent protein wfCGFP2 protein or for such encoding nucleic acids are according to the invention.
Ebenfalls sind Mutanten des fluoreszenten Proteins wfCGFPl Proteins bzw. für solche kodierende Nukleinsäuren erfindungsgemäß.Also mutants of the fluorescent protein wfCGFPl protein or for such encoding nucleic acids according to the invention.
Ebenfalls sind Mutanten des fluoreszenten Proteins wfCGFP2 Proteins bzw. für solche kodierende Nukleinsäuren erfindungsgemäß.Also, mutants of the fluorescent protein wfCGFP2 protein or for such encoding nucleic acids according to the invention.
Die Veränderung von fluoreszenten Proteinen im ähnlichen Bereich der Proteinstruktur sind erfindungsgemäß.The change of fluorescent proteins in the similar region of the protein structure are according to the invention.
Erfindungsgemäß sind die oben genannten mutierten Moleküle, wobei die veränderten physikochemischen und biochemischen Eigenschaften dazu führen, dass sich Zellen stabil mit einem Nukleinsäurekonstrukt transfizieren lassen, welches ein eine Nukleinsäure beinhaltet, die eine solche Mutante kodiert.In accordance with the present invention, the above-mentioned 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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Reportergene für die Technik des „high content Screening" (HCS). HCS steht als Übergriff für moderne Mikroskopie-Technologien zur Zellanalyse. Kennzeichnend für HCS-Verfahren ist die quantitative Erfassung mehrerer Parameter auf zellulärer oder subzellulärer Ebene.The proteins wfCGFPl and wfCGFP2 are suitable as reporter genes for the technique of "high content screening" (HCS) .HCS is an encroachment for modern microscopy technologies for cell analysis.The characteristic feature of HCS methods is the quantitative recording of several parameters at the cellular or subcellular level ,
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Reportergene für zelluläre Systeme speziell für Rezeptoren, für Ionenkanäle, für Transporter, für Transkriptionsfaktoren oder für induzierbare Systeme.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Reportergene in bakteriellen und eukaryotischen Systemen speziell in Säugerzellen, in Bakterien, in Hefen, in Bakulo, in PflanzenThe 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
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Reportergene für zelluläre Systeme in Kombination mit biolumineszenten oder chemolumineszenten Systemen speziell Systemen mit Luziferasen, mit Oxygenasen, mit Phosphatasen.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als sich als Markerproteine, speziell bei der FACS (Fluorescence activated cell sorter) Sortierung. Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Fusionsproteine speziell für Rezeptoren, für Ionenkanäle, für Transporter, für Transkriptionsfaktoren, für Proteinasen, für Kinasen, für Phosphodiesterasen, für Hydrolasen, für Peptidasen, für Transferasen, für Membranproteine, für Glykoproteine.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich zur Immobilisierung speziell durch Antikörper, durch Biotin, durch magnetische oder magnetisierbare Träger.The proteins wfCGFPl and wfCGFP2 are suitable for immobilization, in particular by antibodies, by biotin, by magnetic or magnetizable carriers.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Proteine für Systeme des Energietransfers speziell der FRET- (Fluorescence Resonance Energy Transfer), BRET- (Bioluminescence Resonance Energy Transfer), FET (field effect transistors), FP (fluorescence polarization), HTRF (Homogeneous time-resolved fluorescence) Systemen.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich zur Markierung von Substraten oder Liganden speziell für Proteasen, für Kinasen, für Transferasen, für Transporter, für Ionenkanäle.The proteins wfCGFPl and wfCGFP2 are suitable for labeling substrates or ligands specifically for proteases, for kinases, for transferases, for transporters, for ion channels.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich zur Expression in bakteriellen Sytemen speziell zur Titerbestimmung, als Substrate für biochemische Systeme speziell für Proteinasen und Kinasen.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Marker speziell gekoppelt an Antikörper, gekoppelt an Enzyme, gekoppelt an Rezeptoren, gekoppelt an Ionenkanäle und andere Proteine.The 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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Reportergene bei der pharmakologischen Wirkstoffsuche speziell im HTS (High Throughput Screening).The proteins wfCGFPl and wfCGFP2 are suitable as reporter genes in the pharmacological search for active substances, especially in HTS (High Throughput Screening).
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Komponenten von Detektionssystemen speziell für ELISA (enzyme-linked immunosorbent assay), für Immunohistochemie, für Western- Blot, für die konfokale Mirkoskopie.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Marker für die Analyse von Wechselwirkungen speziell für Protein-Protein-Wechselwirkungen, für DNA-Protein-Wechsel- Wirkungen, für DNA-RNA-Wechselwirkungen, für RNA-RNA- Wechselwirkungen, für RNA- Protein-Wechslewirkungen (DNA : deoxyribonucleic acid; RNA : ribonucleic acid).The 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).
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Marker oder Fusionsproteine für die Expression in transgenen Organismen speziell in Mäusen, in Ratten, in Hamstern und anderen Säugetieren, in Primaten, in Fischen, in Würmern, in Pflanzen. Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Marker oder Fusionsprotein zur Analyse der Embryonalentwicklung.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Marker über einen Kopplungsvermittler speziell über Biotin, über NHS (N-hydroxysulfosuccimide), über CN-Br.The proteins wfCGFPl and wfCGFP2 are useful as markers via a coupling agent specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Reporter gekoppelt an Nukleinsäuren speziell an DNA, an RNA.The proteins wfCGFPl and wfCGFP2 are suitable as reporters coupled to nucleic acids, especially to DNA, to RNA.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Reporter gekoppelt an Proteine oder Peptide.The proteins wfCGFPl and wfCGFP2 are suitable as reporters coupled to proteins or peptides.
Die an Nukleinsäuren oder Peptiden gekoppelten Proteine wfCGFPl sowie wfCGFP2 eignen sich als Sonden speziell für Northern-Blots, für Southern-Blots, für Western-Blots, für ELISA, für Nukleinsäuresequenzierungen, für Proteinanalysen, Chip-Analysen.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als Markierung von pharmakologischen Formulierungen speziell von infektiösen Agenden, von Antikörpern, von „small molecules".The proteins wfCGFPl and wfCGFP2 are suitable as markers of pharmacological formulations especially of infectious agents, of antibodies, of "small molecules".
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich als für geologische Untersuchungen speziell für Meeres-, Grundwasser- und Flussströmungen.The proteins wfCGFPl and wfCGFP2 are suitable for geological investigations especially for ocean, groundwater and river currents.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich zur Expression in Expressionssystemen speziell in in-vitro Translationssystemen, in bakteriellen Systemen, in Hefen Systemen, in Bakulo Systemen, in viralen Systemen, in eukaryotischen Systemen.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.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich zur Visualisierung von Geweben oder Zellen bei chirurgischen Eingriffen speziell bei invasiven, bei nicht-invasiven, bei minimal-invasiven.The proteins wfCGFPl and wfCGFP2 are suitable for the visualization of tissues or cells in surgical interventions, especially in invasive, non-invasive, minimally invasive.
Die Proteine wfCGFPl sowie wfCGFP2 eignen sich auch zur Markierung von Tumorgeweben und anderen phänotypisch veränderten Geweben speziell bei der histologischen Untersuchung, bei operativen Eingriffen.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.
Die Erfindung betrifft auch die Reinigung der Proteine wfCGFPl sowie wfCGFP2 speziell als wildtyp Protein, als Fusionsprotein, als mutagenisiertes Protein.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.
Die Erfindung betrifft auch die Verwendung von wfCGFPl sowie wfCGFP2 auf dem Gebiet der Kosmetik speziell von Badezusätzen, von Lotionen, von Seifen, von Körperfarben, von Zahncreme, von Körperpudern. Die Erfindung betrifft auch die Verwendung von wfCGFPl sowie wfCGFP2 zur Färbung speziell von Nahrungsmitteln, von Badezusätzen, von Tinte, von Textilien, von Kunststoffen.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.
Die Erfindung betrifft auch die Verwendung von wfCGFPl sowie wfCGFP2 zur Färbung von Papier speziell von Grußkarten, von Papierprodukten, von Tapeten, von Bastelartikeln.The invention also relates to the use of wfCGFPl and wfCGFP2 for coloring paper, especially greetings cards, paper products, wallpaper, craft items.
Die Erfindung betrifft auch die Verwendung von wfCGFPl sowie wfCGFP2 zur Färbung von Flüssigkeiten speziell für Wasserpistolen, für Springbrunnen, für Getränke, für Eis.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.
Die Erfindung betrifft auch die Verwendung von wfCGFPl sowie wfCGFP2 zur Herstellung von Spielwaren speziell von Fingerfarbe, von Schminke.The invention also relates to the use of wfCGFPl and wfCGFP2 for the manufacture of toys, especially finger paint, make-up.
Die Erfindung betrifft Organismen, die einen erfindungsgemäßen Vektor enthalten.The invention relates to organisms containing a vector according to the invention.
Die Erfindung betrifft Organismen, die ein erfindungsgemäßes Polypeptid expremieren.The invention relates to organisms expressing a polypeptide of the invention.
Die Erfindung betrifft Organismen, die ein funkionelles Äquivalente von wfCGFPl sowie wfCGFP2 expremieren.The invention relates to organisms expressing a functional equivalent of wfCGFPl and wfCGFP2.
Die Erfindung bezieht sich auf Verfahren zur Expression der erfindungsgemäßen fluoreszierenden Polypeptide in Bakterien, eukaryontischen Zellen oder in in vitro Expressionssystemen.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.
Die Erfindung betrifft auch Verfahren zur Aufreinigung/Isolierung eines erfindungsgemäßen Polypeptides.The invention also relates to methods for purifying / isolating a polypeptide of the invention.
Die Erfindung bezieht sich auf Peptide mit mehr als 5 aufeinanderfolgenden Aminosäuren, die immunologisch durch Antikörper gegen die erfindungsgemäßen fluoreszierende Proteine erkannt werden.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.
Die Erfindung betrifft die Verwendung der erfindungsgemäßen fluoreszierenden Proteine als Marker- und Reportergene, insbesondere für die pharmakologische Wirkstoffsuche und Diagnostik.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.
Die Erfindung betrifft die fluoreszenten Proteine wfCGFPl sowie wfCGFP2 mit den Aminosäuresequenzen repräsentiert durch SEQ ID NO: 2, SEQ ED NO: 6 und der Nukleotidsequenz repräsentiert durch SEQ ID NO: 1 , SEQ ID NO: 5.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.
Die Erfindung betrifft Organismen, die einen erfindungsgemäßen Vektor enthalten.The invention relates to organisms containing a vector according to the invention.
Die Erfindung betrifft Organismen, die ein erfindungsgemäßes Polypeptid expremieren Die Erfindung bezieht sich auf Verfahren zur Expression der erfindungsgemäßen fluoreszierenden Polypeptide in Bakterien, eukaryontischen Zellen oder in in vitro Expressionssystemen.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.
Die Erfindung betrifft auch Verfahren zur Aufreinigung/Isolierung eines erfindungsgemäßen fluoreszierenden Polypeptides.The invention also relates to methods for purification / isolation of a fluorescent polypeptide according to the invention.
Die Erfindung bezieht sich auf Peptide mit mehr als 5 aufeinanderfolgenden Aminosäuren, die immunologisch durch Antikörper gegen die erfmdungsgemäßen fluoreszierende Proteine erkannt werden.The invention relates to peptides having more than 5 consecutive amino acids, which are recognized immunologically by antibodies against the inventive fluorescent proteins.
Die Erfindung betrifft die Verwendung der erfindungsgemäßen fluoreszierenden Proteine als Marker- und Reportergene, insbesondere für die pharmakologische Wirkstoffsuche und Diagnos- tik.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.
Die Erfindung betrifft Organismen, die einen erfindungsgemäßen Vektor enthalten.The invention relates to organisms containing a vector according to the invention.
Die Erfindung betrifft Organismen, die ein erfindungsgemäßes Polypeptid expremierenThe invention relates to organisms expressing a polypeptide of the invention
Die Erfindung bezieht sich auf Verfahren zur Expression der erfϊndungsgemäßen fluoreszierenden Polypeptide in Bakterien, eukaryontischen Zellen oder in in vitro Expressionssystemen.The invention relates to methods for expressing the fluorescent polypeptides according to the invention in bacteria, eukaryotic cells or in in vitro expression systems.
Die Erfindung betrifft auch Verfahren zur Aufreinigung/Isolierung eines erfindungsgemäßen fluoreszierenden Polypeptides.The invention also relates to methods for purification / isolation of a fluorescent polypeptide according to the invention.
Die Erfindung bezieht sich auf Peptide mit mehr als 5 aufeinanderfolgenden Aminosäuren, die immunologisch durch Antikörper gegen die erfindungsgemäßen fluoreszierende Proteine erkannt werden.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.
Die Erfindung betrifft die Verwendung der erfindungsgemäßen fluoreszierenden Proteine als Marker- und Reportergene, insbesondere für die pharmakologische Wirkstoffsuche und Diagnostik.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.
Erfindungsgemäß ist ein fluoreszierendes Protein, dadurch gekennzeichnet, dass seine Sequenz die Sequenz dargestellt in SEQ ID NO: 2 sowie SEQ ID NO: 6 umfasst, sowie funktionelle Fragmente desselben.According to the invention, 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.
Erfindungsgemäß ist des weiteren ein Nukleinsäuremolekül, welches ein Protein kodiert, dass seine Sequenz die Sequenz dargestellt in SEQ K) NO: 2 sowie SEQ ID NO: 6 umfasst, sowie funktionelle Fragmente desselben. Bestandteil der Erfindung ist ein fluoreszierendes Protein, dadurch gekennzeichnet dass es eine Aminosäuresequenz umfasst, welche durch die SEQ ID NO:4 wiedergegeben ist, jedoch im Bereich der Positionen 154 bis 179 eine oder mehrere Mutationen aufweist, welche zu einer schnelleren Faltungszeit des Proteins fuhren, sowie dessen funktionellen Fragmente.Furthermore, according to the invention, 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.
Ebenfalls erfindungsgemäß ist ein Nukleinsäuremolekül, welches ein Protein wie im vorangehenden Abschnitt beschrieben kodiert.Also according to the invention is a nucleic acid molecule which encodes a protein as described in the preceding section.
Erfindungsgemäß ist ein Nukleinsäuremolekül gemäß wie in den vorhergehenden 4 Abschnitten beschrieben, dadurch gekennzeichnet, dass es einen funktionellen Promotor 5' zur kodierenden Sequenz enthält.According to the invention, a nucleic acid molecule as described in the preceding 4 sections, characterized in that it contains a functional promoter 5 'to the coding sequence.
Bestandteil der Erfindung ist ein rekombinanter RNA oder DNA- Vektor, welcher eine Nukleinsäure wie im vorangehenden Abschnitt beschrieben umfasst.A component of the invention is a recombinant RNA or DNA vector which comprises a nucleic acid as described in the preceding section.
Erfindungsgemäß ist ein Organismus, enthaltend einen Vektor wie im vorangehenden Abschnitt beschrieben.According to the invention, an organism containing a vector is as described in the preceding section.
Bestandteil der Erfindung ist ein Verfahren zur Expression eines erfindungsgemäßen PoIy- pepetides in Bakterien, eukaryontischen Zellen, oder in in vitro Translationssystemen.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.
Erfindungsgemäß ist auch ein Verfahren zur Aufreinigung eines wie im vorangehenden Abschnitt beschrieben exprimierten Polypeptides.The invention also provides a method for purifying a polypeptide as described in the preceding section.
Bestandteil der Erfindung ist die Verwendung einer erfindungsgemäßen Nukleinsäure oder 6 als Marker oder Reportergen auch in Kombination mit einem oder mehreren anderen Marker oder Reportergenen.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.
Bestandteil der Erfindung ist ebenfalls die Verwendung eines erfindungsgemäßen Proteins als Marker oder Reportergen auch in Kombination mit einem oder mehreren anderen Marker oder Reportergenproteinen.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.
Ebenfalls erfindungsgemäß ist ein fluoreszierendes Protein, dadurch gekennzeichnet, dass es durch Einfügung von einer oder mehrerer Mutationen eine schnellere Faltungszeit im Vergleich zur ursprünglichen Sequenz aufweist.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.
Die Erfindung betrifft die Verwendung der erfindungsgemäßen fluoreszierenden Proteine als Komponente von Homo- oder Hetero- Di- oder Multimeren fiuoreszenter Proteine, welche direkt oder durch Linker miteinander verbunden sind. Die Erfindung betrifft auch Mutanten des fluoreszenten Proteins CGFP, die zur veränderten Expression des Proteins führen.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.
Die Erfindung betrifft auch Mutanten des fluoreszenten Proteins CGFP, sowie imCGFP und wfCGFP, die eine Etablierung stabiler Zelllinen, die ein fluoreszentes Protein expremieren, ermöglicht. 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.
Nukleotid- und AminosäuresequenzenNucleotide and amino acid sequences
Das mutante fluoreszierende Protein wfCGFPl wird durch die folgende Nukleotidsequenz codiert (SEQ ID NO: 1):The mutant fluorescent protein wfCGFPl is encoded by the following nucleotide sequence (SEQ ID NO: 1):
5'- ATGACTGCACTTACCGAAGGAGCAAAACTGTTCGAGAAAGAAATTCCCTACATTACAG AGTTGGAAGGAGACGTTGAAGGAATGAAATTCATCATCAAAGGTGAAGGTACTGGCG ACGCTACTACTGGCACCATCAAAGCGAAATATATTTGCACAACTGGTGACCTTCCTGT ACCATGGGCTACCATCTTGAGTAGTTTGTCGTATGGTGTTTTCTGTTTCGCTAAGTATC CACGCCACATTGCCGACTTTTTCAAGAGCACACAACCAGATGGTTATTCACAAGACAG AATCATTAGTTTTGACGATGATGGACAATACGACGTCAAAGCCAAGGTTACTTATGAA AACGGAACACTTTATAATAGAGTCACAGTCAAAGGTACTGGCTTCAAATCAAACGGCA ACATCCTTGGTATGAGAGTTCTCTACCATTCACCACCACACGCTGTCTACATCCTTCCT GACCGTAAAAATGGTGGCATTAAAATTGAATACAATAAGGCTTTCGACGTTATGGGCG GTGGTCACCAAATGGCGCGTCACGCCCAATTCAATAAACCACTAGGAGCCTGGGAAG AAGATTATCCGTTGTATCATCATCTTACCGTATGGACTTCTTTCGGAAAAGATCCGGAT GATGATGAAACTGACCATTTGACCATCGTCGAAGTCATCAAAGCTGTTGATTTGGAAA CATACCGTTAA^5 'ATGACTGCACTTACCGAAGGAGCAAAACTGTTCGAGAAAGAAATTCCCTACATTACAG AGTTGGAAGGAGACGTTGAAGGAATGAAATTCATCATCAAAGGTGAAGGTACTGGCG ACGCTACTACTGGCACCATCAAAGCGAAATATATTTGCACAACTGGTGACCTTCCTGT ACCATGGGCTACCATCTTGAGTAGTTTGTCGTATGGTGTTTTCTGTTTCGCTAAGTATC CACGCCACATTGCCGACTTTTTCAAGAGCACACAACCAGATGGTTATTCACAAGACAG AATCATTAGTTTTGACGATGATGGACAATACGACGTCAAAGCCAAGGTTACTTATGAA AACGGAACACTTTATAATAGAGTCACAGTCAAAGGTACTGGCTTCAAATCAAACGGCA ACATCCTTGGTATGAGAGTTCTCTACCATTCACCACCACACGCTGTCTACATCCTTCCT GACCGTAAAAATGGTGGCATTAAAATTGAATACAATAAGGCTTTCGACGTTATGGGCG GTGGTCACCAAATGGCGCGTCACGCCCAATTCAATAAACCACTAGGAGCCTGGGAAG AAGATTATCCGTTGTATCATCATCTTACCGTATGGACTTCTTTCGGAAAAGATCCGGAT GATGATGAAACTGACCATTTGACCATCGTCGAAGTCATCAAAGCTGTTGATTTGGAAA CATACCGTTAA ^
Daraus ergibt sich eine Aminosäuresequenz von (SEQ ID NO: 2):This results in an amino acid sequence of (SEQ ID NO: 2):
MTALTEGAKLFEKEIPYΓΓELEGDVEGMKFΠKGEGTGDATTGTIKAKYICTTGDLPVPWAT ILSSLSYGVFCFAKYPRHIADFFKSTQPDGYSQDRΠSFDDDGQYDVKAKVTYENGTLYNR VTVKGTGFKSNGNILGMRVLYHSPPHAVYILPDRKNGGIKIEYNKAFDVMGGGHQMARH AQFNKPLGAWEEDYPLYHHLTVWTSFGKDPDDDETDHLTIVEVIKAVDLETYRMTALTEGAKLFEKEIPYΓΓELEGDVEGMKFΠKGEGTGDATTGTIKAKYICTTGDLPVPWAT ILSSLSYGVFCFAKYPRHIADFFKSTQPDGYSQDRΠSFDDDGQYDVKAKVTYENGTLYNR VTVKGTGFKSNGNILGMRVLYHSPPHAVYILPDRKNGGIKIEYNKAFDVMGGGHQMARH AQFNKPLGAWEEDYPLYHHLTVWTSFGKDPDDDETDHLTIVEVIKAVDLETYR
Das fluoreszierende Protein CGFP wird durch die folgende Nukleotidsequenz codiert (SEQ E) NO: 3):The fluorescent protein CGFP is encoded by the following nucleotide sequence (SEQ E) NO: 3):
5'-5 '
ATGACTGCACTTACCGAAGGAGCAAAACTGTTCGAGAAAGAAATTCCCTACATTACAG AGTTGGAAGGAGACGTTGAAGGAATGAAATTCATCATCAAAGGTGAAGGTACTGGCG ACGCTACTACTGGCACCATCAAAGCGAAATATATTTGCACAACTGGTGACCTTCCTGT ACCATGGGCTACCATCTTGAGTAGTTTGTCGTATGGTGTTTTCTGTTTCGCTAAGTATC CACGCCACATTGCCGACTTTTTCAAGAGCACACAACCAGATGGTTATTCACAAGACAG AATCATTAGTTTTGACAATGATGGACAATACGATGTCAAAGCCAAGGTTACTTATGAA AACGGAACACTTTATAATAGAGTCACAGTCAAAGGTACTGGCTTCAAATCAAACGGCA ACATCCTTGGTATGAGAGTTCTCTACCATTCACCACCACACGCTGTCTACATCCTTCCT GACCGTAAAAATGGTGGCATGAAAATTGAATACAATAAGGCTTTCGACGTTATGGGCG GTGGTCACCAAATGGCGCGTCACGCCCAATTCAATAAACCACTAGGAGCCTGGGAAG AAGATTATCCGTTGTATCATCATCTTACCGTATGGACTTCTTTCGGAAAAGATCCGGAT GATGATGAAACTGACCATTTGACCATCGTCGAAGTCATCAAAGCTGTTGATTTGGAAA CATACCGTTGA-3\ATGACTGCACTTACCGAAGGAGCAAAACTGTTCGAGAAAGAAATTCCCTACATTACAG AGTTGGAAGGAGACGTTGAAGGAATGAAATTCATCATCAAAGGTGAAGGTACTGGCG ACGCTACTACTGGCACCATCAAAGCGAAATATATTTGCACAACTGGTGACCTTCCTGT ACCATGGGCTACCATCTTGAGTAGTTTGTCGTATGGTGTTTTCTGTTTCGCTAAGTATC CACGCCACATTGCCGACTTTTTCAAGAGCACACAACCAGATGGTTATTCACAAGACAG AATCATTAGTTTTGACAATGATGGACAATACGATGTCAAAGCCAAGGTTACTTATGAA AACGGAACACTTTATAATAGAGTCACAGTCAAAGGTACTGGCTTCAAATCAAACGGCA ACATCCTTGGTATGAGAGTTCTCTACCATTCACCACCACACGCTGTCTACATCCTTCCT GACCGTAAAAATGGTGGCATGAAAATTGAATACAATAAGGCTTTCGACGTTATGGGCG GTGGTCACCAAATGGCGCGTCACGCCCAATTCAATAAACCACTAGGAGCCTGGGAAG AAGATTATCCGTTGTATCATCATCTTACCGTATGGACTTCTTTCGGAAAAGATCCGGAT GATGATGAAACTGACCATTTGACCATCGTCGAAGTCATCAAAGCTGTTGATTTGGAAA CATACCGTTGA-3 \
Daraus ergibt sich eine Aminosäuresequenz von (SEQ ID NO: 4):This results in an amino acid sequence of (SEQ ID NO: 4):
MTALTEGAKLFEKEIPYITELEGDVEGMKFIIKGEGTGDATTGTIK-AKYICTTGDLPVPWATMTALTEGAKLFEKEIPYITELEGDVEGMKFIIKGEGTGDATTGTIK-AKYICTTGDLPVPWAT
ILSSLSYGVFCFAKYPRHIADFFKSTQPDGYSQDRΠSFDNDGQ YDVKAKVTYENGTLYNR VTVKGTGFKSNGNILGMRVLYHSPPHAVYILPDRKNGGMKIEYNKAFDVMGGGHQMAR HAQFNKPLGAWEEDYPLYHHLTVWTSFGKDPDDDETDHLTIVEVΓKAVDLETYRILSSLSYGVFCFAKYPRHIADFFKSTQPDGYSQDRΠSFDNDGQ YDVKAKVTYENGTLYNR VTVKGTGFKSNGNILGMRVLYHSPPHAVYILPDRKNGGMKIEYNKAFDVMGGGHQMAR HAQFNKPLGAWEEDYPLYHHLTVWTSFGKDPDDDETDHLTIVEVΓKAVDLETYR
Das fluoreszierende Protein wfCGFP2 wird durch die folgende Nukleotidsequenz codiert (SEQ ID NO: 5):The fluorescent protein wfCGFP2 is encoded by the following nucleotide sequence (SEQ ID NO: 5):
5Λ- ATGACTGCACTTACCGAAGGAGCAAAACTGTTCGAGAAAGAAATTCCCTACATTACAG AGTTGGAAGGAGACGTTGAAGGAATGAAATTCATCATCAAAGGTGAAGGTACTGGCG ACGCTACTACTGGCACCATCAAAGCGAAATATATTTGCACAACTGGTGACCTTCCTGT ACCATGGGCTACCATCTTGAGTAGTTTGTCGTATGGTGTTTTCTGTTTCGCTAAGTATC CACGCCACATTGCCGACTTTTTCAAGAGCACACAACCAGATGGTTATTCACAAGACAG AATCATTAGTTTTGACGATGATGGACAATACGATGTCAAAGCCAAGGTTACTTATGAA AACGGTACACTTTACAATAGAGTCACAGTCAAAGGTACTGGCTTCAAATCAAACGGCA ACATCCTTGGTATGAGAGTTCTCTACCATTCACCACCACACGCTGTCTACATCCTTCCT GACCGTAAAAATGGTGGCATGAGAATTGAATACAATAAGGCCTTCGACGTTATGGGC GGTGGTCACCAAATGGCGCGTCACGCCCAATTCAATAAACCACTAGGAGCCTGGGAA GAAGATTATCCGTTGTATCATCATCTTACCGTATGGACTTCTTTCGGAAAAGATCCGG ATGATGATGAAACTGACCATTTGACCATCGTCGAAGTCATCAAAGCTGTTGATTTGGA AACATACCGTTAA-3^ 5 Λ - ATGACTGCACTTACCGAAGGAGCAAAACTGTTCGAGAAAGAAATTCCCTACATTACAG AGTTGGAAGGAGACGTTGAAGGAATGAAATTCATCATCAAAGGTGAAGGTACTGGCG ACGCTACTACTGGCACCATCAAAGCGAAATATATTTGCACAACTGGTGACCTTCCTGT ACCATGGGCTACCATCTTGAGTAGTTTGTCGTATGGTGTTTTCTGTTTCGCTAAGTATC CACGCCACATTGCCGACTTTTTCAAGAGCACACAACCAGATGGTTATTCACAAGACAG AATCATTAGTTTTGACGATGATGGACAATACGATGTCAAAGCCAAGGTTACTTATGAA AACGGTACACTTTACAATAGAGTCACAGTCAAAGGTACTGGCTTCAAATCAAACGGCA ACATCCTTGGTATGAGAGTTCTCTACCATTCACCACCACACGCTGTCTACATCCTTCCT GACCGTAAAAATGGTGGCATGAGAATTGAATACAATAAGGCCTTCGACGTTATGGGC GGTGGTCACCAAATGGCGCGTCACGCCCAATTCAATAAACCACTAGGAGCCTGGGAA GAAGATTATCCGTTGTATCATCATCTTACCGTATGGACTTCTTTCGGAAAAGATCCGG ATGATGATGAAACTGACCATTTGACCATCGTCGAAGTCATCAAAGCTGTTGATTTGGA AACATACCGTTAA-3 ^
Daraus ergibt sich eine Aminosäuresequenz von (SEQ ID NO: 6):This results in an amino acid sequence of (SEQ ID NO: 6):
MTALTEGAKLFEKEIPYΠΈLEGDVEGMKFΠKGEGTGDATTGTIKAKYICTTGDLPVPWAT ILSSLSYGVFCFAKYPRHIADFFKSTQPDGYSQDRΠSFDDDGQYDVKAKVTYENGTLYNR VTVKGTGFKSNGNILGMRVLYHSPPHAVYILPDRKNGGMRIEYNKAFDVMGGGHQMAR HAQFNKPLGAWEEDYPLYHHLTVWTSFGKDPDDDETDHLTIVEVIKAVDLETYR Beschreibung der Figuren und TabellenMTALTEGAKLFEKEIPYΠΈLEGDVEGMKFΠKGEGTGDATTGTIKAKYICTTGDLPVPWAT ILSSLSYGVFCFAKYPRHIADFFKSTQPDGYSQDRΠSFDDDGQYDVKAKVTYENGTLYNR VTVKGTGFKSNGNILGMRVLYHSPPHAVYILPDRKNGGMRIEYNKAFDVMGGGHQMAR HAQFNKPLGAWEEDYPLYHHLTVWTSFGKDPDDDETDHLTIVEVIKAVDLETYR Description of the figures and tables
Figur 1FIG. 1
Die Figur 1 zeigt die Plasmidkarte des Vektors pcDNA3-wfCGFPl.FIG. 1 shows the plasmid map of the vector pcDNA3-wfCGFPl.
Figur 2 Die Figur 2 zeigt die Plasmidkarte des Vektors pcDNA3-wfCGFP2.FIG. 2 shows the plasmid map of the vector pcDNA3-wfCGFP2.
Figur 3FIG. 3
Die Figur 3 zeigt die Plasmidkarte des Vektors pcDNA3-CGFP .FIG. 3 shows the plasmid map of the vector pcDNA3-CGFP.
Figur 4FIG. 4
Die Figur 4 zeigt das Alignment von CGFP, wfCGFPl und wfCGFP2 auf Aminosäureebene. (schwarz : CGFP; blau : wfCGFP2, rot : wfCGFPl). Die Positionen 103, 162 und 163 sind schwarz umrandet.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.
Figur 5FIG. 5
Die Figur 5 zeigt das Ergebnis einer FACS-Analyse von wfCGFP2. Links : Messpunkte vor derFigure 5 shows the result of a FACS analysis of wfCGFP2. Left: Measuring points before
Sortierung; Rechts : Messpunkte nach der Sortierung. Fluoreszente Messereignisse in rot.Sorting; Right: Measuring points after sorting. Fluorescent measurement events in red.
Figur 6FIG. 6
Die Figur 6 zeigt ein Foto einer mikroskopischen Aufnahme von stabilen Zellen einer wfCGFP2 expremierenden CHO-Zelllinie.Figure 6 shows a photograph of a micrograph of stable cells of a wfCGFP2 expressing CHO cell line.
Tabelle 1Table 1
Die Tabelle 1 zeigt eine Übersicht über einige fluoreszierende Proteine. Angegeben ist der Name, der Organismus aus dem das Protein isoliert worden ist und die Identifikationsnummer (Acc. No.) des Datenbankeintrages.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.
Tabelle 2Table 2
Die Tabelle 2 zeigt eine Übersicht über einige fluoreszierende Proteine. Angegeben ist der Organismus aus dem das Protein isoliert worden ist, die Anregungs- und Emissionswellenlängen, die bei Spektralanalysen bestimmt worden sind.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.
Tabelle 3Table 3
Die Tabelle 3 zeigt eine Übersicht über einige fluoreszierende Proteine. Angegeben ist der Organismus aus dem das Protein isoliert worden ist, der Name des fluoreszierenden Proteins und eine Auswahl an Patenten bzw. Anmeldungen. BeispieleTable 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
Beispiel 1example 1
Als Vektor zur Herstellung des im folgenden dargestellten Konstruktes wurde das Plasmid pQE7 der Firma Qiagen verwendet. Die Klonierung erfolgte unter Verwendung von molekularbio- logischen Standardmethoden. Die Derivate des Vektors wurde als pQE7-wfCGFPl und pQE7- wfCGFP2 bezeichnet. Die Vektoren pQE7-wfCGFPl und pQE7-wfCGFP2 wurde zur Expression von wfCGFP in bakteriellen Systemen verwendet.As a vector for the preparation of the construct shown below, the plasmid pQE7 from Qiagen was used. 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.
Beispiel 2Example 2
Als Vektor zur Herstellung des im folgenden dargestellten Konstruktes wurde das Plasmid pcDNA3.1(+) der Firma Clontech verwendet. Die Klonierung erfolgte unter Verwendung von molekularbiologischen Standardmethoden.. Das Derivate des Vektors wurde als pcDNA3- wfCGFPl und pcDNA3-wfCGFP2 bezeichnet. Die Vektoren pcDNA3-wfCGFPl und pcDNA3- wfCGFP2 wurde zur Expression von wfCGFP in eukaryotischen Systemen verwendet.As a vector for the preparation of the construct shown below, the plasmid pcDNA3.1 (+) from Clontech was used. 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.
Die Fig. 1 zeigt die Plasmidkarte des Vektors pcDNA3-wfCGFPl. Die Fig. 2 zeigt die Plasmidkarte des Vektors pcDNA3-wfCGFP2. Die Fig. 3 zeigt die Plasmidkarte des Vektors pcDNA3-CGFP.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.
Beispiel 3Example 3
Bakterielle ExpressionBacterial expression
Die bakterielle Expression erfolgte im E. coli Stamm BL21(DE3) durch Transformation der Bakterien mit den Expressionsplasmiden pQE7-wfCGFPl und pQE7-wfCGFP2. Die transformierten Bakterien wurden in LB-Medium bei 37°C für 3 Stunden inkubiert und dieThe 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
Expression für 4 Stunden durch Zugabe von IPTG bis zu einer Endkonzentration von 1 mM induziert. Die induzierten Bakterien wurden durch Zentrifugation geemtet, in PBS resuspendiert und durch Ultraschall aufgeschlossen. Die Fluoreszenz wurde mit Hilfe eines Fluorometers bestimmt.Expression induced for 4 hours by adding IPTG to a final concentration of 1mM. The induced bacteria were harvested by centrifugation, resuspended in PBS and disrupted by ultrasound. The fluorescence was determined by means of a fluorometer.
Beispiel 4Example 4
Eukarvotische ExpressionEukaryotic expression
Die konstitutive eukaryotische Expression erfolgte in CHO-Zellen durch Transfektion der Zellen mit den Expressionsplasmiden pcDNA3 -wfCGFP 1, pcDNA3-wfCGFP2, pcDNA3-CGFP und pcDNA3.1(+) in transienten Experimenten. Hierzu wurden 10000 Zellen pro Loch in DMEM-F 12 Medium auf 96 Loch Mikrotiterplatten plattiert und über Nacht bei 37°C inkubiert. Die Transfektion erfolgte mit Hilfe des Fugene 6 Kits (Roche) nach Herstellerangaben. Die transfizierten Zellen wurden über Nacht bei 37°C in DMEM-F 12 Medium inkubiert. Die Messung der Fluoreszenz erfolgte im Fluorometer bei Raumtemperatur.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. For this purpose, 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.
Zur Herstellung stabiler Zelllinien wurden die transfizierten Zellen mit 2 mg/ml Geneticin selektioniert und die Fluoreszenz der Klone bestimmt.To prepare stable cell lines, the transfected cells were selected with 2 mg / ml geneticin and the fluorescence of the clones was determined.
Beispiel 5Example 5
FACS-AnalvseFACS Analvse
Die konstitutive eukaryotische Expression erfolgte in CHO-Zellen durch Transfektion der Zellen mit den Expressionsplasmiden pcDNA3-wf CGFPl, pcDNA3-wfCGFP2, pcDNA3-CGFP und pcDNA3.1(+). Hierzu wurden CHO Zellen mit Hilfe von Fugene 6 (Firma ROCHE) nach Herstellerangaben transfiziert und in DMEM-F 12 Medium über Nacht bei 37°C inkubiert. Nach 24 bzw. 48 Stunden wurden die Zellen durch Acutase (Firma Invitrogene; Durchführung nach Herstellerangaben) abgelöst, durch Zentrifugation peletiert und anschließend in PBS + 0,5 % BSA aufgenommen. Die FACS Sortierung erfolgte mit dem Gerät FACSCalibur (Firma Becton Dickinson) nach Herstellerangaben. Insgesamt wurden 9xlOE7 Zellen sortiert. Die eluierten Zellen wurden durch Zentrifugation peletiert, in PBS aufgenommen und in Zellkulturflaschen überführt. Zur Herstellung stabiler Zelllinien wurden die sortierten Zellen mit 2 mg/ml Geneticin selektioniert und die Fluoreszenz der Klone bestimmt.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 (+). For this purpose, 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.
Die Fig. 5 zeigt das Ergebnis der FACS-Analyse in einer grafischen Darstellung.FIG. 5 shows the result of the FACS analysis in a graphical representation.
Beispiel 6Example 6
Sequenzvergleichsequence comparison
Um die Sequenzen der fluoreszenten Proteine CGFP, wfCGFPl und wfCGFP2 vergleichen und darstellen zu können, wurde ein Alignment der Aminosäuresequenzen durchgeführt.In order to be able to compare and display the sequences of the fluorescent proteins CGFP, wfCGFPl and wfCGFP2, an alignment of the amino acid sequences was carried out.
Die Fig. 4 zeigt das Alignment der fluoreszenten Proteien CGFP, wfCGFPl und wfCGFP2 auf Aminosäureebene. Literatur / PatenteFIG. 4 shows the alignment of the fluorescent proteins CGFP, wfCGFPl and wfCGFP2 at the amino acid level. Literature / Patents
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US patent no. 5,874,304US Pat. No. 5,874,304
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US patent no. 6,020,192US Pat. No. 6,020,192
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Claims

Patentansprüche claims
1. Ein fluoreszierendes Protein, dadurch gekennzeichnet, dass seine Sequenz die Sequenz dargestellt in SEQ E) NO: 2 oder 6 umfasst, sowie funktionelle Fragmente desselben.1. A fluorescent protein, characterized in that its sequence comprises the sequence shown in SEQ E) NO: 2 or 6, as well as functional fragments thereof.
2. Ein Nukleinsäuremolekül, welches ein Protein gemäß Anspruch 1 kodiert.2. A nucleic acid molecule which encodes a protein according to claim 1.
3. Ein fluoreszierendes Protein, dadurch gekennzeichnet dass es eine Aminosäuresequenz umfasst, welche durch die SEQ ED NO:4 wiedergegeben ist, jedoch im Bereich der Positionen 153 bis 173, sowie 93 bis 112 eine oder mehrere Mutationen aufweist, welche zu einer veränderten Expression des Proteins führen, sowie dessen funktionellen Fragmente.3. A fluorescent protein, 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 153 to 173, and 93 to 112, which leads to an altered expression of the Protein as well as its functional fragments.
4. Ein Nukleinsäuremolekül, welches ein Protein gemäß Anspruch 3 kodiert.4. A nucleic acid molecule which encodes a protein according to claim 3.
5. Ein Nukleinsäuremolekül gemäß Ansprüchen 2 oder 4, dadurch gekennzeichnet, dass es einen funktionellen Promotor 5' zur kodierenden Sequenz enthält.5. A nucleic acid molecule according to claims 2 or 4, characterized in that it contains a functional promoter 5 'to the coding sequence.
6. Rekombinanter RNA oder DNA- Vektor, welcher eine Nukleinsäure gemäß Anspruch 5 umfasst.6. Recombinant RNA or DNA vector comprising a nucleic acid according to claim 5.
7. Nicht humaner Organismus, enthaltend einen Vektor gemäß Anspruch 6.7. Non-human organism containing a vector according to claim 6.
8. Verfahren zur Expression eines Polypeptides gemäß Anspruch 1 oder 3 in Bakterien, eukaryontischen Zellen, oder in in vitro Translationssystemen.8. A method for expression of a polypeptide according to claim 1 or 3 in bacteria, eukaryotic cells, or in in vitro translation systems.
9. Verfahren zur Aufreinigung eines gemäß Anspruch 8 exprimierten Polypeptides.9. A process for the purification of a polypeptide expressed according to claim 8.
10. Verwendung einer Nukleinsäure gemäß Anspruch 2, 3, 5 oder 6 als Marker oder Reportergen.10. Use of a nucleic acid according to claim 2, 3, 5 or 6 as a marker or reporter gene.
11. Verwendung einer Nukleinsäure gemäß Anspruch 2, 3, 5, oder 6 als Marker oder Reportergen in Kombination mit einem oder mehreren anderen Marker oder Reportergenen.11. Use of a nucleic acid according to claim 2, 3, 5, or 6 as a marker or reporter gene in combination with one or more other marker or reporter genes.
12. Verwendung eines Proteins gemäß Anspruch 1 oder 3 als Marker oder Reportergen.12. Use of a protein according to claim 1 or 3 as a marker or reporter gene.
13. Verwendung eines Proteins gemäß Anspruch 1 oder 3 als Marker oder Reportergen in Kombination mit einem oder mehreren anderen Marker oder Reportergenproteinen. 13. Use of a protein according to claim 1 or 3 as a marker or reporter gene in combination with one or more other markers or reporter gene proteins.
14. Ein fluoreszierendes Protein, dadurch gekennzeichnet, dass es durch Einfügung von einer oder mehrerer Mutationen eine veränderte Expression im Vergleich zur ursprünglichen Sequenz aufweist.14. A fluorescent protein, characterized in that it has an altered expression compared to the original sequence by insertion of one or more mutations.
15. Ein fluoresziendes Protein, dadurch gekennzeichnet, dass es durch Einfügung von einer oder mehrerer Mutationen die Etablierung stabil expremierender Zelllinien ermöglicht.15. A fluorescent protein, characterized in that it allows by introducing one or more mutations, the establishment of stable expressing cell lines.
16. Zelllinie, dadurch gekennzeichnet, dass sie mit einer Nukleinsäure, welche eine Nukleinsäure gemäß Ansprüchen 2, 4, 5 oder 6 beinhaltet, stabil transfiziert ist.Cell line, characterized in that it is stably transfected with a nucleic acid comprising a nucleic acid according to claims 2, 4, 5 or 6.
17. Verwendung einer Zelllinie gemäß Anspruch 16 in einem Testsystem in der pharmazeutischen Wirkstoffforschung zur Identifizierung neuer Wirkstoffe. 17. Use of a cell line according to claim 16 in a test system in pharmaceutical drug discovery for the identification of new drugs.
PCT/EP2007/004967 2006-06-07 2007-06-05 FLUORESCENT PROTEINS wfCGFP, and use thereof WO2007140983A1 (en)

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