Classifications

• • 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
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
• • 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

Definitions

• the invention relates to the photoprotein AQdecay, its nucleotide and amino acid sequence, as well as the activity and use of the photoprotein AQdecay.
• Bioluminescence is the phenomenon of light generation by living beings. It is the result of biochemical reactions in cells, in which the chemical energy is released in the form of light quanta (so-called cold emission by chemiluminescence). Such generated light is monochromatic because it is emitted at a discrete electron transition, but may be shifted to longer wavelength spectral regions by secondary luminescent dyes (e.g., fluorescent jellyfish of the genus Aequoria).
• secondary luminescent dyes e.g., fluorescent jellyfish of the genus Aequoria
• the biological function is manifold: in the depth of the sea between 200 and 1000 m (mesopelagic), around 90% of all living beings light up.
• the light signals are used here for partner advertising, deception and bait. Even fireflies and fireflies use the light signals to find a partner.
• the importance of lighting bacteria, fungi and unicellular algae, however, is unclear. It is believed that it is responsible for the coordination of many
• Single individuals of a large population is used or represents a kind of biological clock.
• a variety of coelenterates is bioluminescent (Moria et al., 1974). These organisms emit blue or green light.
• the aequorin from Aequoria victoria (Shimomura et al., 1969), identified as the first light-producing protein in 1962, emitted a blue light and non-green light as an isolated protein, as observed phenotypically in Aequoria victoria.
• the green fluorescent protein could be isolated from Aequoria victoria, which makes the medusa phenotypically green due to the stimulation by the aequorin (Johnson et al., 1962, Hastings et al., 1969, Inouye et al., 1994).
• Clytin Inouye et al., 1993
• mitrocomin Fagan et al., 1993
• obelin obelin
• Table 2 Overview of some photoproteins. Given are the organism from which the protein has been isolated, the name of the photoprotein and a selection of patents or applications.
• Bioluminescence is widely used in the art today, e.g. in the form of bio-indicators for environmental pollution or in biochemistry for the sensitive detection of proteins, for
• Photoproteins differ not only in their nucleotide and amino acid sequence, but also in their biochemical and physical properties. It has been shown that altering the amino acid sequence of photoproteins can alter their physical and biochemical properties. Examples of mutagenized photoproteins are described in the literature (US 6,495,355, US 5,541,309, US 5,093,240, Shimomura et al., 1986).
• 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.
• the most common reporter genes include beta-galactosidase (Alam et al., 1990), alkaline phosphatase (Yang et al., 1997, Cullen et al., 1992), luciferases and other photoproteins (Shinomura, 1985, Phillips GN, 1997; Snowdowne et al., 1984).
• Luminescence 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 results in 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.
• Amino acids have altered spectral or biochemical properties. These include obelin W92F (Vysotski et al., 2003) and aequorin (Shrestha et al., 2002; Ohmiya et al., 1993).
• the aequorin mutant AQdecay shows a temporally altered release of light compared to the photoprotein aequorin or other photoproteins.
• the mutation at position 139 responsible for the temporal change in light release was combined with a mutation at position 89.
• the change at position 89 has already been described and leads to a change in the spectral properties of the photoprotein.
• the selected combination shows, in addition to the temporally altered release of light, also changed spectral properties.
• a combination of replacing other amino acids with the change at position 139 is possible. Also the combination of
• the photoprotein AQdecay surprisingly shows a hitherto not yet described slowed kinetics of the release of light or luminescence. This property allows the use of the photoprotein in addition to the usual uses specifically for the
• the invention relates to the photoprotein AQdecay 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: 2.
• the invention also relates to functional equivalents of AQdecay.
• Functional equivalents are those proteins that have comparable physicochemical properties.
• the invention relates to aequorin photoproteins which have one or more amino acid mutations in the region of the amino acid positions 129-149, 124-134, preferably 137-141, in particular 138-140 (based on GenBank # AAA27716), which lead to an altered properties of the bioluminescence. Furthermore, the invention relates to aequorin photoproteins which have an amino acid mutation in the position 139 (based on GenBank # AAA27716), which lead to altered properties of the bioluminescence. Aequorin photoproteins may also be those photoproteins which have a similar motif in the region of amino acids 134-145 as the truncated aequorin (GenBank # AAA27716). As regions with a similar Motif here are those sequences that have an identity of 80%, preferably 90% in this area.
• the invention relates to combinations of aequorin photoproteins in the range of amino acid positions 79-99, 84-94, preferably 87-91, in particular 88-90 (based on GenBank
• the invention relates to combinations of aequorin photoproteins which have an amino acid mutation at position 89 (based on GenBank # AAA27716) which leads to an altered spectrum of fluorescence or bioluminescence, with mutations in the region of amino acid position 139.
• photoproteins which have a maximum in the fluorescence or bioluminescence spectrum in the range of 480-520 nm, preferably 485-515 nm, particularly preferably in the range from 490-510 nm, 495 to 505, or in particular at 500 nm.
• Aequorin photoproteins may also be those photoproteins which have a similar motif in the region of amino acids 84-94 as the truncated one
• Aequorin (GenBank # AAA27716). 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 AQdecay protein or for such encoding nucleic acids are according to the invention.
• the photoprotein AQdecay is particularly suitable as a reporter gene for cellular systems
• Receptors for ion channels, for transporters, for transcription factors or for inducible systems.
• the photoprotein AQdecay is also useful as a reporter gene by labeling, identification and characterization of cell organelles specific to mitochondria.
• the photoprotein from AQdecay is also suitable as a reporter gene for the determination of parameters inside and outside of cell organelles, especially of mitochondria, especially of calcium concentrations.
• the photoprotein AQdecay is suitable as a reporter gene in bacterial and eukaryotic systems, especially in mammalian cells, in bacteria, in yeasts, in bacculo, in plants.
• the photoprotein AQdecay is suitable as a reporter gene for cellular systems in combination with bioluminescent or chemiluminescent systems, especially systems with luciferases, with oxygenases, with phosphatases.
• the photoprotein AQdecay is particularly suitable as a fusion protein for receptors, ion channels, transporters, transcription factors, proteinases, kinases, phosphodiesterases, hydrolases, peptidases, transferases, membrane proteins and glycoproteins.
• the photoprotein AQdecay is suitable for immobilization especially by antibodies, by biotin, by magnetic or magnetizable carriers.
• the photoprotein AQdecay is suitable as protein for systems of energy transfer especially the fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), field effect transistor (FET), fluorescence polarization (FPRF), homogenous time-resolved (HTRF) fluorescence) systems.
• FRET fluorescence resonance energy transfer
• BRET bioluminescence resonance energy transfer
• FET field effect transistor
• FPRF fluorescence polarization
• HTRF homogenous time-resolved fluorescence
• the photoprotein AQdecay is suitable as a marker of substrates or ligands especially for proteases, for kinases, for transferases.
• the photoprotein AQdecay is suitable for expression in bacterial systems especially for titer determination, as a substrate for biochemical systems especially for proteinases and kinases.
• the photoprotein AQdecay is useful as a marker specifically coupled to antibodies coupled to enzymes coupled to receptors coupled to ion channels and other proteins.
• the photoprotein AQdecay is suitable as a reporter gene for pharmacological drug discovery, especially in HTS (High Throughput Screening).
• the photoprotein AQdecay is useful as a reporter gene in the characterization, identification and analysis of ion channels, especially of the type p2x, TRP, SCN, KCN, CNG, ACCN.
• the photoprotein AQdecay is suitable as a component of detection systems especially for ELISA (enzyme-linked immunosorbent assay), for immunohistochemistry, for Western blot, for confocal microscopy.
• ELISA enzyme-linked immunosorbent assay
• the photoprotein AQdecay is useful as a marker for the analysis of interactions specifically for protein-protein interactions, for DNA-protein interactions, for DNA-RNA interactions, for RNA-RNA interactions, for RNA-protein interactions (DNA: deoxyribonucleic acid; RNA: ribonucleic acid;).
• the photoprotein AQdecay is useful as a marker or fusion protein for expression in transgenic organisms, especially in mice, in rats, in hamsters and other mammals, in primates, in fish, in worms, in plants.
• the photoprotein AQdecay is useful as a marker or fusion protein for the analysis of embryonic development.
• the photoprotein AQdecay is suitable as a marker via a coupling agent specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
• the photoprotein AQdecay is suitable as a reporter coupled to nucleic acids, especially to DNA, to RNA.
• the photoprotein AQdecay is suitable as a reporter coupled to proteins or peptides.
• the photoprotein AQdecay is suitable as a reporter for the measurement of intracellular or extracellular calcium concentrations.
• the photoprotein AQdecay is suitable for the characterization of signal cascades in cellular systems.
• the coupled to nucleic acids or peptides photoprotein AQdecay is particularly suitable as a probe for Northern blots, for Southern blots, for Western blots, for ELISA, for nucleic acid sequencing, for protein analysis, chip analyzes.
• the photoprotein AQdecay is suitable for the labeling of pharmacological formulations especially of infectious agents, of antibodies, of "small molecules".
• the photoprotein AQdecay is suitable for geological investigations especially for ocean, groundwater and river currents.
• the photoprotein AQdecay is suitable for expression in expression systems, especially in vitro
• the photoprotein AQdecay is suitable for the visualization of tissues or cells during surgery, especially in invasive, non-invasive, minimally invasive.
• the photoprotein AQdecay is also suitable for the marking of tumor tissues and other phenotypically altered tissues, especially during histological examination, during surgical procedures.
• the invention also relates to the purification of the photoprotein AQdecay specifically as a wild-type protein, as a fusion protein, as a mutagenized protein.
• the photoprotein AQdecay is suitable for the simultaneous measurement of different reporter genes in an expression system (multiplexing).
• the invention also relates to the use of the photoprotein AQdecay in the field of cosmetics, especially bath preparations, lotions, soaps, body colors, toothpaste, body powders.
• the invention also relates to the use of the photoprotein AQdecay for staining specifically
• the invention also relates to the use of the photoprotein AQdecay for coloring paper, especially greetings cards, paper products, wallpaper, craft items.
• the invention also relates to the use of the photoprotein AQdecay for dyeing liquids especially for water pistols, for fountains, for drinks, for ice cream.
• the invention also relates to the use of the photoprotein AQdecay for the manufacture of toys especially of finger paint, make-up.
• the invention relates to nucleic acid molecules which encode the polypeptide disclosed by SEQ ID NO: 2 or functional equivalents or functional fragments thereof.
• the invention further relates to nucleic acid molecules or functional equivalents or functional fragments thereof, selected from the group consisting of
• nucleic acid molecules encoding a polypeptide comprising the amino acid sequence disclosed by SEQ ID NO: 2;
• nucleic acid molecules whose complementary strand hybridizes with a nucleic acid molecule from a) or b) under stringent conditions and whose expression product has the biological function of a photoprotein;
• a stringent hybridization of nucleic acid molecules is carried out in an aqueous
• the invention relates to the abovementioned nucleic acid molecules in which the sequence contains a functional promoter 5 ⁇ to the photoprotein-encoding sequence or the sequence encoding the leader or signal sequence.
• the invention also relates to nucleic acid molecules as described above that are part of recombinant DNA or RNA vectors.
• the invention relates to organisms containing such a vector.
• the invention relates to photoproteins which are encoded by the nucleotide sequences described above.
• the invention relates to methods for expressing the photoprotein polypeptides according to the invention in bacteria, eukaryotic cells or in in vitro expression systems.
• the invention also relates to methods for the purification / isolation of a photoprotein polypeptide according to the invention.
• the invention relates to the use of the erf ⁇ ndungshielen, coding for photoproteins
• Nucleic acids as marker or reporter genes, in particular for pharmacological drug discovery and diagnostics.
• the invention relates to the use of the photoproteins according to the invention or a nucleic acid according to the invention which codes for a photoprotein as marker or reporter or as marker or reporter gene.
• the invention relates to the use of the photoprotein AQdecay (SEQ ID NO: 2) or its functional fragments or equivalents or the use of a coding for the photoprotein AQdecay nucleic acid or its functional fragments or equivalents as a marker or reporter or as a marker or reporter gene in particular for the pharmacological drug discovery and diagnostics.
• the invention relates to the use of the nucleic acid shown in SEQ ID NO: 1 as a marker or reporter gene, in particular for the pharmacological drug discovery and diagnostics.
• the invention also relates to polyclonal or monoclonal antibodies which recognize a polypeptide according to the invention.
• the invention also relates to monoclonal or polyclonal antibodies containing the photoprotein
• the invention also relates to a nucleic acid as described in the preceding paragraphs which contains a functional promoter 5 ⁇ to the coding sequence.
• the invention includes recombinant DNA or RNA vectors containing the nucleic acids described above.
• Organisms containing a vector as described above are also erf ⁇ ndungshiel.
• a polypeptide encoded by a nucleic acid sequence as described above is also part of the invention. Also according to the invention is a method for the expression of the aforementioned polypeptides in bacteria, eukaryotic cells or in in vitro expression systems.
• a component of the invention is likewise a process for the purification / isolation of a polypeptide according to the invention.
• the invention relates to the use of a nucleic acid according to the invention as marker or
• the invention also relates to the use of a photoprotein according to the invention as a marker or reporter.
• polyppeptide according to the invention in combination with one or more luciferases and / or one or more photoproteins.
• a photoprotein or a functional fragment thereof which has one or more mutations in the range of 129-149, 124-134, preferably 137-141, in particular 138-140 (based on GenBank # AAA27716) and which has an altered, specifically slowed bioluminescence signal having.
• nucleic acid molecule which contains a sequence which codes for a protein according to the two preceding sections.
• a further component of the invention is a process for the preparation of a photoprotein, characterized in that in a photoprotein in the region defined by position 129-149, 124-134, preferably 137-141, in particular 138-140 referred to GenBank # AAA27716 one or several mutations are introduced, resulting in a change in bioluminescence.
• a photoprotein prepared by a method as described in the preceding section is also according to the invention.
• the invention also relates to other photoproteins which have altered kinetics of light release by one or more changes in the amino acid sequence.
• the invention also addresses the use of other altered photoproteins for the described uses of the photoprotein AQdecay.
• Photoproteins with altered kinetics of light release are particularly suitable as reporter genes in cell-based methods, especially in pharmacological drug discovery and characterization, especially in diagnostics.
• Photoproteins with altered kinetics of light release, particularly a slower release of light or a prolonged period of time in which light is released, are particularly useful in the study of ion channels.
• the invention also relates to codon-optimized variants of the proteins according to the invention for altering the biochemical or physicochemical properties, especially the improved expression, especially the altered stability.
• the invention also relates to fusions of erfindungsgenä built proteins with recognition peptides for the transport or localization of the proteins of the invention in cell organelles or
• the invention also relates to variants of the proteins according to the invention which lead to a change in the spectral properties, the luminescence intensity, the substrate specificity, the use of cofactors, the calcium affinity or other physicochemical or biochemical properties.
• Expression is the production of a molecule which, after introduction of the gene into a suitable host cell, permits the transcription and translation of the foreign gene cloned into an expression vector.
• Expression vectors contain the control signals required for the expression of genes in cells of prokaryotes or eukaryotes.
• expression vectors can be constructed in two different ways.
• transcriptional fusion the protein encoded by the cloned foreign gene is synthesized as an authentic, biologically active protein.
• the expression vector carries all the 5 'and 3' control signals required for expression.
• the protein encoded by the cloned foreign gene is expressed as a hybrid protein together with another protein that is easily detected.
• the 5 'and 3' control signals required for the expression, including the start codon, and possibly a part of the sequences coding for the N-terminal regions of the hybrid protein to be formed are derived from the vector.
• the additional introduced protein portion not only in many cases stabilizes the protein encoded by the cloned foreign gene from degradation by cellular
• Proteases can also be used to detect and isolate the resulting hybrid protein deploy.
• Expression can be transient as well as stable. Suitable host organisms are bacteria, yeasts, viruses as well as eukaryotic systems.
• Protein purification involves a variety of established methods and procedures
• Solid-liquid separation is a basic operation in protein isolation. Both in the separation of the cells from the culture medium and in the clarification of the crude extract after cell disruption and removal of cell debris, in the separation of precipitates after precipitation, etc., the process step is required. It is done by centrifugation and
• the cell wall must be destroyed or rendered permeable.
• high pressure homogenizers or stirred ball or glass bead mills are used.
• mechanical cell integrations and ultrasound treatment are used.
• Extracellular proteins accumulate in relatively dilute solutions. They must be concentrated as well as extracellular proteins prior to their further use. In addition to the already mentioned methods, ultrafiltration has also proven itself - even on an industrial scale. Inorganic salts as concomitants of proteins are often undesirable for specific applications. They can be removed, inter alia, by gel filtration, dialysis and diafusion.
• the photoprotein AQdecay is encoded by the following nucleotide sequence (SEQ ID NO: 1):
• the photoprotein aequorin (Genbank: AAA27716) has the following amino acid sequence (SEQ ID NO: 7). Positions 89 and 139 are in bold and underlined.
• Fig. 1 shows the plasmid map of the vector pET22b-AQdecay.
• Fig. 2 shows the plasmid map of the vector pcDNA3-AQdecay
• FIG. 3 shows the result of the eukaryotic expression of AQdecay in CHO cells.
• the experimental procedure was carried out as described in Example 4.
• Fig. 4 shows the result of the bacterial expression of AQdecay.
• the insertion of the cDNA was carried out in the interface Ndel / Xhol of the vector pET22b (Novagen).
• the vector was named pET22b AQdecay.
• Fig. 1 shows the plasmid map of the vector pET22b-AQdecay.
• the plasmid pcDNA3.1 (+) from Clontech was used as a vector for the preparation of the construct shown below.
• the derivative of the vector was termed pcDNA3-AQdecay.
• the vector pcDNA3-AQdecay was used to express AQdecay in eukaryotic systems.
• Fig. 2 shows the plasmid map of the vector pcDNA3-AQdecay.
• Bacterial expression was carried out in E. coli by transformation of the bacteria with the expression plasmids pET22b-AQdecay.
• the transformed bacteria were incubated in LB medium at 37 0 C for 3 hours and the expression according to the manufacturer (Novagen) induced.
• the induced bacteria were harvested by centrifugation, in 50 mM Tris / HCl (pH 9.0) + 5 mM
• Luminometer measured. The integration time of the measurement was 40 seconds.
• FIG. 4 shows the kinetics of the bioluminescence measurement of AQdecay in bacteria.
• the constitutive eukaryotic expression was carried out in CHO cells by transfecting the cells with the expression plasmids pcDNA3-AQdecay 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 0 C in DMEM F12 medium. Subsequently, the medium was removed and replaced with 50 ⁇ l of coelenterazine (10E-07 M coelenterazine in PBS).
• the cells were incubated for 24 hours at 28 ° C and then added ATP (adenosine triphosphate) to a final concentration of 1 uM.
• ATP adenosine triphosphate
• the measurement was started immediately after the addition in the luminometer.
• the integration time was 1 second, with a total measurement time of 60 seconds.
• Fig. 3 shows the results of the bioluminescence measurement of AQdecay in CHO cells.
• FIG. 5 shows the kinetics of the bioluminescence measurement of AQdecay in CHO cells
• FIG. 7 shows the alignment of AQdecay with aequorin (wildtype; wt) at the amino acid level.
• E. coli BL21 (DE3) transformed with pET22b AQdecay or pET22b (without integrated cDNA).
• the culture and digestion of the bacteria was carried out as described in Example 3.
• the measurement data was collected for a period of 60 seconds with a 1 second integration time.
• Figure 4 shows the results of the kinetic analysis of AQdecay in bacteria.
• CHO Choinese hamster ovarian cells
• pcDNA3 -AQdecay pcDNA3 (without integrated cDNA). Transfection and measurement were carried out as described in Example 4.
• Measurement data was collected for a period of 60 seconds with a 1 second integration time.
• Figure 5 shows the results of the kinetic analysis of AQdecay in CHO cells.
• the photoprotein Aqdecay is useful as a component of multiplexing readout methods in which multiple reporter genes (e.g., luciferases or photoproteins) are used in an experimental approach.
• multiple reporter genes e.g., luciferases or photoproteins
• CHO cells expressing the wild-type aequorin.
• the cell expressing the wild-type aequorin additionally expressed a G-protein coupled receptor (eg Neuromedin U Receptor 2).
• the cell mixture was spread on 96, 384 or 1536 well microtiter plates and incubated for 24 hours at 37 0 C.
• G-protein receptor agonist leads to intracellular calcium release, which can be read by the wild-type aequorin (light release by wild-type aequorin).
• an agonist that activates a CHO endogenous receptor eg, ATP
• activates the AQdecay of the second cell type e.g, ATP
• the photoprotein AQdecay or its equivalents is suitable for fusion with peptides, leader sequences, translocation signals, proteins or protein fragments for transport or localization in specific cell compartments or organelles.
• the photoprotein according to the invention was fused with the peptide MSVLTPLLLRGLTGSARRLPVPRAKIHSLPPEGKL. Fusion of the peptide to the amino acid sequence of AQdecay results in translocation of the fusion protein into the mitochondria of the eukaryotic host cell.
• the mitochondrial localized photoprotein AQdecay can be used to measure calcium concentration within the
• Mitochondria are used.
• the fusion of the described peptide before the amino acid sequence of the AQdecay photoprotein was carried out at the nucleic acid level using standard molecular biological methods.
• Literature / Patents
• Green Fluorescent Protein Properties, Applications, and Protocols (Chalfie, M. and Kain, S., eds) pp. 45-70. Wiley-Liss, Inc.

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  • 2005-05-13 DE DE102005022146A patent/DE102005022146A1/de not_active Withdrawn
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