EP1638998A1 - Isolated photoprotein bolinopsin, and the use thereof - Google Patents

Isolated photoprotein bolinopsin, and the use thereof

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Publication number
EP1638998A1
EP1638998A1 EP04740054A EP04740054A EP1638998A1 EP 1638998 A1 EP1638998 A1 EP 1638998A1 EP 04740054 A EP04740054 A EP 04740054A EP 04740054 A EP04740054 A EP 04740054A EP 1638998 A1 EP1638998 A1 EP 1638998A1
Authority
EP
European Patent Office
Prior art keywords
photoprotein
nucleic acid
bolinopsin
sequence
acid molecules
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP04740054A
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German (de)
French (fr)
Inventor
Stefan Golz
Svetlana Markova
Ludmila Burakova
Ludmila Frank
Eugene Vysotski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer AG
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Bayer Healthcare AG
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Publication date
Application filed by Bayer Healthcare AG filed Critical Bayer Healthcare AG
Publication of EP1638998A1 publication Critical patent/EP1638998A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the invention relates to the photoprotein bolinopsin, its nucleotide and amino acid sequence, and the activity and use of the photoprotein bolinopsin.
  • Bioluminescence is the phenomenon of light generation by living things. 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 through chemiluminescence). Light generated in this way is monochromatic, because it is emitted at a discrete electron transition, but can be shifted into longer-wave spectral ranges by secondary fluorescent dyes (e.g. fluorescent proteins in light jellyfish of the genus Aequora).
  • secondary fluorescent dyes e.g. fluorescent proteins in light jellyfish of the genus Aequora
  • the biological function is multifaceted: around 90% of all living things shine in the sea depth between 200 and 1000 m (mesopelagial).
  • the light signals are used here for partner advertising, deception and as bait. Fireflies and fireflies also use the light signals to find partners.
  • a large number of coelenterates are bioluminescent (JMorin et al., 1974). These organisms emit blue or green light.
  • the aequorin from Aequoria victoria identified as the first light-producing protein in 1962 (Shimomura et al., 1969) emitted a blue light and not green light as phenotypically observed in Aequoria victoria.
  • the green fluorescent protein (GFP) was later isolated from Aequoria victoria, which due to the excitation by the Aequorin makes the medusa appear phenotypically green (Johnson et al, 1962; Hastings et al., 1969; Inouye et al, 1994).
  • Clytin Inouye et al., 1993
  • Mitrocomin Fluorescence Activated protein
  • Obelin Ularionov et al., 1995
  • Bioluminescence is widely used in technology today, e.g. in the form of bio-indicators for environmental pollution or in biochemistry for the sensitive detection of proteins, for the quantification of certain compounds or as so-called “reporters” in the study of cellular gene regulation.
  • the photoproteins differ not only because of their nucleotide and amino acid sequence, but also because of their biochemical and physical properties. It was shown that changing the amino acid sequence of photoproteins can change the 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 referred to as genes whose gene products can be easily detected using simple biochemical or histochemical methods. There are at least 2 types of reporter genes.
  • Resistance genes are genes whose expression gives a cell resistance to antibiotics or other substances, the presence of which in the growth medium leads to cell death if the resistance gene is missing.
  • Reporter genes are used in genetic engineering as merged 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; Snow Downe et. al., 1984).
  • Luminescence is the radiation 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 the ground state. If this reaction is catalyzed by an enzyme, one speaks of bioluminescence.
  • the enzymes involved in the reaction are generally referred to as luciferases.
  • reaction products were incubated for 30 minutes at 37 ° C. with proteinase K and the cDNA was precipitated with ethanol.
  • the expression cDNA bank was carried out using the “SMART cDNA Library Construction Kit” from Clontech (USA) according to the manufacturer's instructions.
  • the cloning was carried out into the expression vector pTriplEx2 (Clontech; USA).
  • the expression vectors were electroplated into bacteria of strain E. coli XLl-Blue transformed.
  • the bacteria were plated on LB culture media and incubated for 24 hours at 37 ° C. Replica plating was then carried out by transferring the bacteria to a further culture medium plate using a nitrocellulose filter. The replica plate was again incubated for 24 hours at 37 ° C. and the grown bacterial colonies were transferred to LB liquid medium. After the addition of JPTG (final concentration 0.1 mM), the bacteria were incubated for 4 hours at 37 ° C. on a shaker. The bacteria were harvested by centrifugation and the bacterial mass was resuspended in 0.5 ml digestion buffer (5 mM EDTA, 20 mM Tris-HCL pH 9.0) at 0 ° C. The bacteria were then disrupted using ultrasound.
  • JPTG final concentration 0.1 mM
  • the lysates were incubated at 4.degree. C. for 3 hours after the addition of coelenterazine (final concentration 10E-07 M). The bioluminescence was then measured after the addition of calcium chloride (final concentration 20 mM) in the luminometer.
  • a photoprotein was identified.
  • the photoprotein was called bolinopsin.
  • the photoprotein bolinopsin is shown in detail below.
  • the photoprotein bolinopsin shows the highest homology at the amino acid level to aequorin from Aequoria victoria with an identity of 44% (shown in Example 8; FIG. 7). At the nucleic acid level, the identity is below 30% (shown in Example 9; FIG. 6).
  • the BLAST method was used for sequence comparison (Altschul et al., 1997).
  • the invention also relates to functional equivalents of bolinopsin.
  • Functional equivalents are those proteins which have comparable physicochemical properties and are at least 70% homologous to SEQ LD NO: 2. A homology of at least 80% or 90% is preferred. A homology of at least 95% is particularly preferred.
  • the photoprotein bolinopsin is suitable as a reporter gene for cellular systems, especially for receptors, for ion channels, for transporters, for transcription factors or for inducible systems.
  • the photoprotein bolinopsin is suitable as a reporter gene in bacterial and eukaryotic systems, especially in mammalian cells, in bacteria, in yeast, in Bakulo, in plants.
  • the photoprotein bolinopsin is suitable as reporter genes for cellular systems in combination with bioluminescent or chemiluminescent systems, especially systems with luciferases, with oxygenases, with phosphatases.
  • the photoprotein bolinopsin is suitable as a fusion protein especially for receptors, for ion channels, for transporters, for transcription factors, for proteinases, for kinases, for phosphodiesterases, for hydrolases, for peptidases, for transferases, for membrane proteins, for glycoproteins.
  • the photoprotein bolinopsin is particularly suitable for immobilization by antibodies, by biotin, by magnetic or magnetizable carriers.
  • the photoprotein bolinopsin is suitable as a protein for energy transfer systems, in particular FRET (fluorescence resonance energy transfer), BRET (bioluminescence resonance energy transfer), FET (field effect transistors), FP (fluorescence polarization), FJLTRF (homogeneous time-resolved) fluorescence) systems.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • FET field effect transistors
  • FP fluorescence polarization
  • FJLTRF homogeneous time-resolved fluorescence
  • the photoprotein bolinopsin is suitable as a label for substrates or J ligands, especially for proteases, for kinases, for transferases.
  • the photoprotein bolinopsin is suitable for expression in bacterial systems especially for titer determination, as a substrate for biochemical systems especially for proteinases and kinases.
  • the photoprotein bolinopsin is particularly suitable as a marker coupled to antibodies, coupled to enzymes, coupled to receptors, coupled to ion channels and other proteins.
  • the photoprotein bolinopsin is suitable as a reporter gene for pharmacological drug discovery, especially in HTS (high throughput screening).
  • the photoprotein bolinopsin 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 bolinopsin is suitable as a marker 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 photoprotein bolinopsin is suitable 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 bolinopsin is suitable as a marker or fusion protein for analyzing embryonic development.
  • the photoprotein bolinopsin is suitable as a marker via a coupling mediator, specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
  • the photoprotein bolinopsin is suitable as a reporter coupled to nucleic acids, especially to DNA, to RNA.
  • the photoprotein bolinopsin is suitable as a reporter coupled to proteins or peptides.
  • the photoprotein bolinopsin is suitable as a reporter for measuring intra- or extracellular calcium concentrations.
  • the photoprotein bolinopsin is suitable for the characterization of signal cascades in cellular systems.
  • the photoprotein bolinopsin coupled to nucleic acids or peptides 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 analysis.
  • the photoprotein bolinopsin is suitable for labeling pharmacological formulations, especially infectious agents, antibodies, and “small molecules”.
  • the photoprotein 33olinopsin is suitable for geological investigations especially for ocean, groundwater and river currents.
  • the photoprotein bolinopsin is 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 photoprotein bolinopsin is suitable for the visualization of tissues or cells during surgical interventions, especially for invasive, non-invasive, and minimally invasive.
  • the photoprotein bolinopsin is also suitable for marking tumor tissues and other phenotypically modified tissues, especially for histological examinations and surgical interventions.
  • the invention also relates to the purification of the photoprotein bolinopsin, especially as a wild-type protein, as a fusion protein, as a mutagenized protein.
  • the invention also relates to the use of the photoprotein bolinopsin in the field of cosmetics, in particular bath additives, lotions, soaps, body colors, toothpaste, body powders.
  • the invention also relates to the use of the photoprotein bolinopsin for coloring food, bath additives, ink, textiles and plastics.
  • the invention also relates to the use of the photoprotein bolinopsin for coloring paper, especially greeting cards, paper products, wallpapers, and handicrafts.
  • the invention also relates to the use of the photoprotein bolinopsin for coloring liquids, especially for water pistols, for fountains, for drinks, for ice.
  • the invention also relates to the use of the photoprotein bolinopsin for the production of toys, especially of finger paint, of make-up.
  • the invention relates to nucleic acid molecules which encode the polypeptide by SEQ LD NO: 2.
  • the invention relates to the polypeptide with the amino acid sequence disclosed in SEQ LD NO: 2.
  • the invention further relates to nucleic acid molecules selected from the group consisting of
  • nucleic acid molecules encoding a polypeptide which comprises 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 which have the biological function of a photoprotein;
  • nucleic acid molecules which differ from those mentioned under c) due to the degeneracy of the genetic code
  • nucleic acid molecules which have a sequence homology of at least 95% to SEQ LD NO: 1 and whose protein product has the biological function of a photoprotein;
  • nucleic acid molecules which have a sequence homology of at least 65% to SEQ ID NO: 1 and whose protein product has the biological function of a photoprotein.
  • the invention also relates to nucleic acid molecules which have a sequence hiiology of at least 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60% to SEQ ID NO: 1 and code for a polypeptide which has the properties of a photoprotein.
  • the invention relates to the above-mentioned nucleic acid molecules in which the sequence contains a functional promoter 5 "to the sequence coding for the photoprotein.
  • the invention also relates to nucleic acid molecules as described above, which are part of recombinant DNA or RNA vectors.
  • the invention relates to organisms which contain such a vector.
  • the invention relates to oligonucleotides with more than 10 consecutive nucleotides which are identical or complementary to the DNA or RNA sequence of the bolinopsin molecules or of the further molecules according to the invention.
  • 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 purifying / isolating a photoprotein polypeptide according to the invention.
  • the invention relates to peptides with more than 5 consecutive amino acids which are recognized immunologically by antibodies against the photoprotems according to the invention.
  • the invention relates to the use of the nucleic acids according to the invention, coding for photoproteins, as marker or reporter genes, in particular for pharmacological search for active substances and diagnostics.
  • the invention relates to the use of the photoprotems according to the invention or to a nucleic acid according to the invention coding for a photoprotein as a marker or reporter or as a marker or reporter gene.
  • the invention relates to the use of the photoprotein bolinopsin. (SEQ JJD NO: 2) or the use of an Nvxidein Textre coding for the photoprotein bolinopsin as a marker or reporter or as a marker or reporter gene, in particular for 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 pharmacological drug search 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 which recognize the photoprotein bolinopsin (SEQ ID NO: 2).
  • Expression refers to the production of a molecule which, after the gene has been introduced into a suitable host cell, allows 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.
  • transcription fusions the protein encoded by the cloned-in foreign gene is called authentic, biologically active protein synthesized.
  • the expression vector carries all 5 and 3 control signals required for expression.
  • the protein encoded by the cloned-in foreign gene is expressed as a hybrid protein together with another protein that can be easily detected.
  • the additional protein part introduced not only stabilizes the protein encoded by the cloned-in foreign gene in many cases before it is broken down by cellular proteases, but can also be used for the detection and isolation of the hybrid protein formed. Expression can be both transient and stable. Both bacteria, yeasts, viruses and eukaryotic systems are suitable as host organisms.
  • protein purification The isolation of proteins (even after overexpression) is often referred to as protein purification.
  • a variety of established methods and processes are available for protein purification.
  • Solid-liquid separation is a basic operation in protein isolation.
  • the process step is required 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 the cell debris, in the separation of precipitates after precipitation, etc. It is done by centrifugation and filtration.
  • the cell wall must be destroyed or made permeable by obtaining intracellular proteins.
  • high-pressure homogenizers or agitator ball or glass bead mills are used.
  • mechanical cell integrations and ultrasound treatment are used.
  • Extracellular proteins are obtained in relatively dilute solutions. Like extracellular proteins, they must be concentrated before further use. In addition to the processes already mentioned, ultrafiltration has also proven itself - also on an industrial scale.
  • Inorganic salts as accompanying substances in proteins are often undesirable for specific applications. Among other things, you can removed by gel filtration, dialysis and diafiltration.
  • Numerous proteins are used as dry preparations. Vacuum, freeze and spray drying are important drying methods.
  • the photoprotein bolinopsin is encoded by the following nucleotide sequence (SEQ ID NO: 1):
  • Fig. 1 shows the plasmid map of the vector pTriplEX2-bolinopsin.
  • Fig. 2 shows the plasmid map of the vector pcDNT A3 -Bolinopsin
  • Fig. 3 shows the exitation of bolinopsin.
  • Y intensity
  • X wavelength [um].
  • Fig. 4 shows the fluorescence of bolinopsin.
  • Y intensity
  • X wavelength [nm].
  • Fig. 5 shows the bioluminescence of bolinopsin.
  • Y intensity;
  • X wavelength [nm].
  • Fig. 6 shows the alignment of bolinopsin and aequorin (aequoria victoria) at the nucleic acid level.
  • Fig. 7 shows the alignment of bolinopsin and aequorin (aequoria victoria) at the amino acid level.
  • Fig. 8 shows the result of the bioluminescence measurement of bolinopsin after bacterial expression.
  • Y luminescence in RLU [relative light units];
  • FIG. 9 shows the result of the bioluminescence measurement of bolinopsin after bacterial expression as a function of the coelenterazine derivative used.
  • Y luminescence in RLU [relative light units];
  • the plasmid pTriplEx2 from Clontech was used as the vector for producing the construct shown below.
  • the derivative of the vector was named pTriplEx2- 5 bolinopsin.
  • the vector pTriplEx2-bolinopsin was used to express bolinospin in bacterial systems.
  • 1 shows the plasmid map of the vector pTriplEX2-bolinopsin.
  • the plasmidO pcDNA3.1 (+) from Clontech was used to produce the construct shown below.
  • the derivative of the vector was named pcDNA3-bolinopsin.
  • the vector pcDNA3-bolinopsin was used to express bolinospin in eukaryotic systems.
  • Bacterial expression took place in the E. coli strain BL21 (DE3). by transforming the bacteria with the expression plasmids pTriplEX2-bolinopsin and pTriplEX2. The transformed bacteria were incubated in LB medium at 37 ° C. for 3 hours and expression was induced for 4 hours by adding IPTG to a final concentration of 1 mM. The O-induced bacteria were harvested by centrifugation, resuspended in PBS + 5 mM EDTA and disrupted by ultrasound. The lysate was incubated with coelenterazine in the dark for 3 hours. Immediately after the addition of 5 mM calcium chloride, the bioluminescence was measured in the luminometer. The integration time of the measurement was 40 seconds.
  • the constitutive eukaryotic expression was carried out in CHO cells by transfection of the cells with the expression plasmids pcDNA3-bolinopsin and pcDNA3.1 (+) in transient experiments. instruments. For this purpose, 10,000 cells per well were plated in DMEM-F12 medium on 96 well microtiter plates and incubated at 37 ° C. overnight. The transfection was carried out using the Fugene 6 kit (Röche) according to the manufacturer's instructions. The transfected cells were incubated overnight at 37 ° C in DMEM-F12 medium.
  • FIG. 6 shows the alignment of bolinopsin with aequorin (Aequoria victoria) at the nucleic acid level.
  • E. coli BL21 (DE3) were transformed with the plasmids pTriplEX2-CGFP and pTriplEX2.
  • the induction was carried out by adding 1 nmM IPTG and incubating for 4 hours at 37 ° C.
  • the bacteria were then harvested and resuspended in PBS.
  • the lysis was carried out by ultrasound.
  • the fluorescence or bioluminescence was then measured. The maximum of the excitation was 352 nm, the fluorescence at 452 nm and that of the bioluminescence at 468 nm.
  • FIG. 3 shows the exitation of bolinopsin.
  • FIG. 4 shows the fluorescence of bolinopsin 5 shows the bioluminescence of bolinopsin
  • FIG. 9 shows the coelenterazine derivatives as potential substrates for bolinopsin and a graphic representation of the luminescence measurement for 30 seconds at 8.7 kV in the luminometer (RLU, relative light units).

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Abstract

The invention relates to the photoprotein bolinopsin, to the nucleotide and amino acids sequence and to the activity thereof, and to use of the photoprotein bolinopsin.

Description

Isoliertes Photoprotein Bolinopsin, sowie dessen VerwendungIsolated photoprotein bolinopsin and its use
Die Erfindung betrifft das Photoprotein Bolinopsin, dessen Nukleotid- und Aminosäuresequenz, sowie die Aktivität und Verwendung des Photoproteins Bolinopsin.The invention relates to the photoprotein bolinopsin, its nucleotide and amino acid sequence, and the activity and use of the photoprotein bolinopsin.
PhotoproteinePhoto proteins
Als Biolumineszenz bezeichnet man das Phänomen der Lichterzeugung durch Lebewesen. Sie ist das Ergebnis von biochemischen Reaktionen in Zellen, bei denen die chemische Energie in Form von Lichtquanten abgegeben wird (sog. kalte Emission durch Chemolumineszenz). Derartig erzeugtes Licht ist monochromatisch, denn es wird bei einem diskreten Elektronen-Übergang abgestrahlt, kann aber durch sekundäre Leuchtfarbstoffe (z.B. fluoreszierende Proteine bei Leucht- quallen der Gattung Aequora) in längerwellige Spektralbereiche verschoben werden.Bioluminescence is the phenomenon of light generation by living things. 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 through chemiluminescence). Light generated in this way is monochromatic, because it is emitted at a discrete electron transition, but can be shifted into longer-wave spectral ranges by secondary fluorescent dyes (e.g. fluorescent proteins in light jellyfish of the genus Aequora).
Die biologische Funktion ist vielfältig: In der Meerestiefe zwischen 200 und 1000 m (Meso- pelagial) leuchten rund 90% aller Lebewesen. Die Leuchtsignale werden hier zur Partnerwerbung, Täuschung und als Köder eingesetzt. Auch Glühwürmchen und Leuchtkäfer nutzen die Lichtsignale zur Partnersuche. Die Bedeutung des Leuchtens von Bakterien, Pilzen und einzelligen Algen ist dagegen unklar. Es wird vermutet, dass es zur Koordination von vielen Einzel-Individuen einer großen Population eingesetzt wird oder eine Art biologische Uhr darstellt.The biological function is multifaceted: around 90% of all living things shine in the sea depth between 200 and 1000 m (mesopelagial). The light signals are used here for partner advertising, deception and as bait. Fireflies and fireflies also use the light signals to find partners. The importance of the glowing of bacteria, fungi and unicellular algae, however, is unclear. It is believed that it is used to coordinate many individual individuals in a large population or is a kind of biological clock.
Eine Vielzahl an Coelenteraten ist biolumineszent (JMorin et al., 1974). Diese Organismen emittieren blaues oder grünes Licht. Das 1962 als erstes licht produzierendes Protein identifizierte Aequorin aus Aequoria victoria (Shimomura et al., 1969) emittierte als isoliertes Protein ein blaues Licht und nicht grünes Licht wie phänotypisch beobachtet bei Aequoria victoria. Später konnte das grün fluoreszierende Protein (GFP) aus Aequoria victoria isoliert werden, das aufgrund der Anregung durch das Aequorin die Meduse phänotypisch grün erscheinen lässt (Johnson et al, 1962; Hastings et al., 1969; Inouye et al, 1994). Als weitere Photoproteine konnten noch Clytin (Inouye et al., 1993), Mitrocomin (Fagan et al., 1993) und Obelin (Ularionov et al., 1995) identifiziert und beschrieben werden. Tabelle 1: Übersicht über einige Photoproteine. Angegeben ist der Name, der Organismus aus dem das Protein isoliert worden ist und die Identifikationsnummer (Acc. No.) des Datenbankeintrages .A large number of coelenterates are bioluminescent (JMorin 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 (Shimomura et al., 1969) emitted a blue light and not green light as phenotypically observed in Aequoria victoria. The green fluorescent protein (GFP) was later isolated from Aequoria victoria, which due to the excitation by the Aequorin makes the medusa appear phenotypically green (Johnson et al, 1962; Hastings et al., 1969; Inouye et al, 1994). Clytin (Inouye et al., 1993), Mitrocomin (Fagan et al., 1993) and Obelin (Ularionov et al., 1995) were identified and described as further photoproteins. Table 1: Overview of some photoproteins. The name, the organism from which the protein has been isolated and the identification number (Acc. No.) of the database entry are given.
Tabelle 2; Übersicht über einige Photoproteine. Angegeben ist der Organismus aus dem das Protein isoliert worden ist, der Name des Photoproteins und eine Auswahl an Patenten bzw. Anmeldungen.Table 2; Overview of some photoproteins. The organism from which the protein has been isolated, the name of the photoprotein and a selection of patents or applications are indicated.
Biolumineszenz wird heute in der Technik vielfältig genutzt, z.B. in Form von Bio-Indikatoren für Umweltverschmutzung oder in der Biochemie zum empfindlichen Nachweis von Proteinen, zur Quantifizierung bestimmter Verbindungen oder als sogenannte "Reporter" bei der Untersuchung zellulärer Gen-Regulation.Bioluminescence is widely used in technology today, e.g. in the form of bio-indicators for environmental pollution or in biochemistry for the sensitive detection of proteins, for the quantification of certain compounds or as so-called "reporters" in the study of cellular gene regulation.
Die Photoproteine unterscheiden sich nicht nur aufgrund ihrer Nukleotid- und Aminosäuresequenz, sondern auch aufgrund ihrer biochemischen und physikalischen Eigenschaften. Es konnte gezeigt werden, dass durch die Veränderung der Aminosauresequenz von Photo- proteinen die physikalischen und biochemischen Eigenschaften verändert werden können. Beispiele von mutagenisierten Photoproteinen sind in der Literatur beschrieben (US 6,495,355; US 5,541,309; US 5,093,240; Shimomura et al., 1986).The photoproteins differ not only because of their nucleotide and amino acid sequence, but also because of their biochemical and physical properties. It was shown that changing the amino acid sequence of photoproteins can change the 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).
Die Lichterzeugung durch die oben genannten Photoproteine erfolgt durch die Oxidation von Coelenterazin (Haddock et al., 2001; Jones et al., 1999).The light generation by the above-mentioned photoproteins takes place through the oxidation of coelenterazine (Haddock et al., 2001; Jones et al., 1999).
Reportersystemereporter systems
Als Reporter- oder Indikatorgen 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 referred to as genes whose gene products can be easily detected using simple biochemical or histochemical methods. There are at least 2 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 gives a cell resistance to antibiotics or other substances, the presence of which in the growth medium leads to cell death if the resistance gene is missing.
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 Phosphatase (Yang et al., 1997; Cullen et al., 1992), Luciferasen und andere Photoproteine (Shinomura, 1985; Phillips GN, 1997; Snowdowne et.al., 1984).2. Reporter genes. Reporter gene products are used in genetic engineering as merged 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; Snow Downe 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 is the radiation 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 Biolümineszenz. Die an der Reaktion beteiligten Enzyme werden generell als Luziferasen bezeichnet.A 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 the ground state. If this reaction is catalyzed by an enzyme, one speaks of bioluminescence. The enzymes involved in the reaction are generally referred to as luciferases.
Einordung der Spezies Bolinopsis infundibulumClassification of the Bolinopsis infundibulum species
Eumetazoa → Radiata → Ctenophora → Tentaculata → Lobata → Bolinopsidae → Bolinopsis infundibulum Isolierung der cDNAEumetazoa → Radiata → Ctenophora → Tentaculata → Lobata → Bolinopsidae → Bolinopsis infundibulum Isolation of the cDNA
Zur Untersuchung der Biolumineszenz-Aktivität der Spezies Bolinopsis infundibulum wurden Exemplare im Weißen Meer (Biologische Station Kartesh, Russland) gefangen und in flüssigem Stickstoff gelagert. Zur Erstellung der cDNA-Bibliotheken von Bolinopsis infundibulum, wurde die poly(a)+ RNA mit Hilfe des „Straight A" Isolationsmethode von Novagen (USA) isoliert.To investigate the bioluminescence activity of the species Bolinopsis infundibulum, specimens were caught in the White Sea (Kartesh Biological Station, Russia) and stored in liquid nitrogen. To create the Bolinopsis infundibulum cDNA libraries, the poly (a) + RNA was isolated using the "Straight A" isolation method from Novagen (USA).
Zur Herstellung der cDNA wurde eine RT-PCR durchgeführt. Hierzu wurden 1 μg RNA mit Reverser Transkriptase (Superscribt Gold II) nach folgendem Schema inkubiert:An RT-PCR was carried out to produce the cDNA. For this purpose, 1 μg RNA was incubated with reverse transcriptase (Superscript Gold II) according to the following scheme:
PCR 1. 30 Sekunden 95°C 2. 6 Minuten 68°C 3. 10 Sekunden 95°C 4. 6 Minuten 68°C 17 Zyklen von Schritt 4 nach Schritt 3PCR 1. 30 seconds 95 ° C 2. 6 minutes 68 ° C 3. 10 seconds 95 ° C 4. 6 minutes 68 ° C 17 cycles from step 4 to step 3
Die Reaktionsprodukte wurden zur Inaktivierung der Polymerase für 30 Minuten bei 37°C mit Proteinase K inkubiert und die cDNA mit Ethanol präzipitiert. Die Expression-cDNA Bank wurde mit Hilfe des „SMART cDNA Library Construction Kits" der Firma Clontech (USA) nach Herstellerangaben durchgeführt. Die Klonierung erfolgte in den Expressionsvektor pTriplEx2 (Clontech; USA). Die Expressionsvektoren wurden durch Elektroporation in Bakterien des Stammes E. coli XLl-Blue transformiert.To inactivate the polymerase, the reaction products were incubated for 30 minutes at 37 ° C. with proteinase K and the cDNA was precipitated with ethanol. The expression cDNA bank was carried out using the “SMART cDNA Library Construction Kit” from Clontech (USA) according to the manufacturer's instructions. The cloning was carried out into the expression vector pTriplEx2 (Clontech; USA). The expression vectors were electroplated into bacteria of strain E. coli XLl-Blue transformed.
Die Bakterien wurden auf LB-Nährböden plattiert und für 24 Stunden bei 37°C inkubiert. An- schließend wurde eine Replikaplattierung durchgeführt, indem die Bakterien mit Hilfe eines Nitro- cellulosefilters auf eine weitere Nährbodenplatte übertragen wurde. Die Replikaplatte wurde wiederum für 24 Stunden bei 37°C inkubiert und die gewachsenen Bakterienkolonien in LB- Flüssigmedium übertragen. Nach der Zugabe von JPTG (Endkonzentration 0,1 mM) wurden die Bakterien für 4 Stunden bei 37°C auf einem Schüttler inkubiert. Die Bakterien wurden durch Zentrifugation geerntet und die Bakterienmasse in 0,5 ml Aufschlusspuffer (5 mM EDTA, 20 mM Tris-HCL pH 9,0) bei 0°C resuspendiert. Anschließend erfolgte der Aufschluss der Bakterien durch Ultraschall.The bacteria were plated on LB culture media and incubated for 24 hours at 37 ° C. Replica plating was then carried out by transferring the bacteria to a further culture medium plate using a nitrocellulose filter. The replica plate was again incubated for 24 hours at 37 ° C. and the grown bacterial colonies were transferred to LB liquid medium. After the addition of JPTG (final concentration 0.1 mM), the bacteria were incubated for 4 hours at 37 ° C. on a shaker. The bacteria were harvested by centrifugation and the bacterial mass was resuspended in 0.5 ml digestion buffer (5 mM EDTA, 20 mM Tris-HCL pH 9.0) at 0 ° C. The bacteria were then disrupted using ultrasound.
Die Lysate wurden nach der Zugabe von Cöelenterazine (Endkonzentration 10E-07 M) bei 4°C für 3 Stunden inkubiert. Anschließend erfolgte die Messung der Biolumineszenz nach der Zugabe von Calziumchlorid (Endkonzentration 20 mM) im Luminometer.The lysates were incubated at 4.degree. C. for 3 hours after the addition of coelenterazine (final concentration 10E-07 M). The bioluminescence was then measured after the addition of calcium chloride (final concentration 20 mM) in the luminometer.
Es wurde ein Photoprotein identifiziert. Das Photoprotein wurde als Bolinopsin bezeichnet. Im Folgenden wird das Photoprotein Bolinopsin im einzelnen dargestellt. BolinopsinA photoprotein was identified. The photoprotein was called bolinopsin. The photoprotein bolinopsin is shown in detail below. Bolinopsin
Das Photoprotein Bolinopsin zeigt die höchste Homologie auf Aminosäureebene zu Aequorin aus Aequoria victoria mit einer Identität von 44 % (gezeigt in Beispiel 8; Fig. 7). Auf Nukleinsäure- ebene liegt die Identität unter 30 % (gezeigt in Beispiel 9; Fig. 6). Zum Sequenzvergleich wurde das BLAST-Verfahren verwendet (Altschul et al., 1997).The photoprotein bolinopsin shows the highest homology at the amino acid level to aequorin from Aequoria victoria with an identity of 44% (shown in Example 8; FIG. 7). At the nucleic acid level, the identity is below 30% (shown in Example 9; FIG. 6). The BLAST method was used for sequence comparison (Altschul et al., 1997).
Die Erfindung betrifft auch funktioneile Äquivalente von Bolinopsin. Fun tionelle Äquivalente sind solche Proteine, die vergleichbare physikochemische Eigenschaften haben und mindestens 70 % homolog sind zu SEQ LD NO: 2. Bevorzugt ist eine Homologie von mindestens 80 % oder 90 %. Besonders bevorzugt ist eine Homologie von mindestens 95 %.The invention also relates to functional equivalents of bolinopsin. Functional equivalents are those proteins which have comparable physicochemical properties and are at least 70% homologous to SEQ LD NO: 2. A homology of at least 80% or 90% is preferred. A homology of at least 95% is particularly preferred.
Das Photoprotein Bolinopsin eignet sich als Reportergen für zelluläre Systeme speziell für Rezeptoren, für Ionenkanäle, für Transporter, für Transkriptionsfaktoren oder für induzierbare Systeme.The photoprotein bolinopsin is suitable as a reporter gene for cellular systems, especially for receptors, for ion channels, for transporters, for transcription factors or for inducible systems.
Das Photoprotein Bolinopsin eignet sich als Reportergen in bakteriellen und eukaryotischen Systemen speziell in Säugerzellen, in Bakterien, in Hefen, in Bakulo, in Pflanzen.The photoprotein bolinopsin is suitable as a reporter gene in bacterial and eukaryotic systems, especially in mammalian cells, in bacteria, in yeast, in Bakulo, in plants.
Das Photoprotein Bolinopsin eignet sich als Reportergene für zelluläre Systeme in Kombination mit biolumineszenten oder chemolumineszenten Systemen speziell Systemen mit Luziferasen, mit Oxygenasen, mit Phosphatasen.The photoprotein bolinopsin is suitable as reporter genes for cellular systems in combination with bioluminescent or chemiluminescent systems, especially systems with luciferases, with oxygenases, with phosphatases.
Das Photoprotein Bolinopsin eignet sich als Fusionsprotein speziell für Rezeptoren, für Ionenkanäle, für Transporter, für Transkriptionsfaktoren, für Proteinasen, für Kinasen, für Phospho- diesterasen, für Hydrolasen, für Peptidasen, für Transferasen, für Membranproteine, für Glykoproteine.The photoprotein bolinopsin is suitable as a fusion protein especially for receptors, for ion channels, for transporters, for transcription factors, for proteinases, for kinases, for phosphodiesterases, for hydrolases, for peptidases, for transferases, for membrane proteins, for glycoproteins.
Das Photoprotein Bolinopsin eignet sich zur Immobilisierung speziell durch Antikörper, durch Biotin, durch magnetische oder magnetisierbare Träger.The photoprotein bolinopsin is particularly suitable for immobilization by antibodies, by biotin, by magnetic or magnetizable carriers.
Das Photoprotein Bolinopsin eignet sich als Protein für Systeme des Energietransfers speziell der FRET- (Fluorescence Resonance Energy Transfer), BRET- (Bioluminescence Resonance Energy Transfer), FET (field effect transistors), FP (fluorescence polarization), FJLTRF (Homogeneous time-resolved fluorescence) Systemen.The photoprotein bolinopsin is suitable as a protein for energy transfer systems, in particular FRET (fluorescence resonance energy transfer), BRET (bioluminescence resonance energy transfer), FET (field effect transistors), FP (fluorescence polarization), FJLTRF (homogeneous time-resolved) fluorescence) systems.
Das Photoprotein Bolinopsin eignet sich als Markierung von Substraten oder JLiganden speziell für Proteasen, für Kinasen, für Transferasen.The photoprotein bolinopsin is suitable as a label for substrates or J ligands, especially for proteases, for kinases, for transferases.
Das Photoprotein Bolinopsin eignet sich zur Expression in bakteriellen Sytemen speziell zur Titer- bestimmung, als Substrat für biochemische Systeme speziell für Proteinasen und Kinasen. Das Photoprotein Bolinopsin eignet sich als Marker speziell gekoppelt an Antikörper, gekoppelt an Enzyme, gekoppelt an Rezeptoren, gekoppelt an Ionenkanäle und andere Proteine.The photoprotein bolinopsin is suitable for expression in bacterial systems especially for titer determination, as a substrate for biochemical systems especially for proteinases and kinases. The photoprotein bolinopsin is particularly suitable as a marker coupled to antibodies, coupled to enzymes, coupled to receptors, coupled to ion channels and other proteins.
Das Photoprotein Bolinopsin eignet sich als Reportergen bei der pharmakologischen Wirkstoffsuche speziell im HTS (High Throughput Screening).The photoprotein bolinopsin is suitable as a reporter gene for pharmacological drug discovery, especially in HTS (high throughput screening).
Das Photoprotein Bolinopsin eignet sich als Komponente von Detektionssystemen speziell für ELISA (enzyme-linked immunosorbent assay), für Irnmunohistochemie, für Western-Blot, für die konfokale Mikroskopie.The photoprotein bolinopsin is suitable as a component of detection systems especially for ELISA (enzyme-linked immunosorbent assay), for immunohistochemistry, for Western blot, for confocal microscopy.
Das Photoprotein Bolinopsin eignet sich als Marker für die Analyse von Wechselwirkungen speziell für Protein-Protein- Wechselwirkungen, für DNA-Protein-Wechselwirkungen, für DNA- RNA-Wechselwirkungen, für RNA-RNA- Wechselwirkungen, für RNA-Protein-Wechsel- wirkungen (DNA : deoxyribonucleic acid; RNA : ribonucleic acid; ).The photoprotein bolinopsin is suitable as a marker 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;).
Das Photoprotein Bolinopsin eignet sich als Marker oder Fusion. sprotein für die Expression in transgenen Organismen speziell in Mäusen, in Ratten, in Hamstern und anderen Säugetieren, in Primaten, in Fischen, in Würmern, in Pflanzen.The photoprotein bolinopsin is suitable 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.
Das Photoprotein Bolinopsin eignet sich als Marker oder Fusionsprotein zur Analyse der Embryonalentwicklung.The photoprotein bolinopsin is suitable as a marker or fusion protein for analyzing embryonic development.
Das Photoprotein Bolinopsin eignet sich als Marker über einen Kopplungs Vermittler speziell über Biotin, über NHS (N-hydroxysulfosuccimide), über CN-Br.The photoprotein bolinopsin is suitable as a marker via a coupling mediator, specifically via biotin, via NHS (N-hydroxysulfosuccimide), via CN-Br.
Das Photoprotein Bolinopsin eignet sich als Reporter gekoppelt an Nukleinsäuren speziell an DNA, an RNA.The photoprotein bolinopsin is suitable as a reporter coupled to nucleic acids, especially to DNA, to RNA.
Das Photoprotein Bolinopsin eignet sich als Reporter gekoppelt an Proteine oder Peptide.The photoprotein bolinopsin is suitable as a reporter coupled to proteins or peptides.
Das Photoprotein Bolinopsin eignet sich als Reporter zur Messung von intra- oder extrazellulären Calziumkonzentrationen.The photoprotein bolinopsin is suitable as a reporter for measuring intra- or extracellular calcium concentrations.
Das Photoprotein Bolinopsin eignet sich zur Charakterisierung von Signalkaskaden in zellulären Systemen.The photoprotein bolinopsin is suitable for the characterization of signal cascades in cellular systems.
Das an Nukleinsäuren oder Peptiden gekoppelte Photoprotein Bolinopsin eignet sich als Sonde speziell für Northern-Blots, für Southern-Blots, für Western-Blots, für ELISA, für Nuklein- säuresequenzierungen, für Proteinanalysen, Chip-Analysen. Das Photoprotein Bolinopsin eignet sich Markierung von pharmakologischen Formulierungen speziell von infektiösen Agentien, von Antikörpern, von „small molecules".The photoprotein bolinopsin coupled to nucleic acids or peptides 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 analysis. The photoprotein bolinopsin is suitable for labeling pharmacological formulations, especially infectious agents, antibodies, and “small molecules”.
Das Photoprotein 33olinopsin eignet sich für geologische Untersuchungen speziell für Meeres-, Grundwasser- und Flussströmungen.The photoprotein 33olinopsin is suitable for geological investigations especially for ocean, groundwater and river currents.
Das Photoprotein Bolinopsin eignet sich zur Expression in Expressionssystemen speziell in in- vitro Translationssystemen, in bakteriellen Systemen, in Hefe Systemen, in Bakulo Systemen, in viralen Systemen, in eukaryotischen Systemen.The photoprotein bolinopsin is 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.
Das Photoprotein Bolinopsin eignet sich zur Visualisierung von Geweben oder Zellen bei chirurgischen Eingriffen speziell bei invasiven, bei nicht-invasiven, bei minimal-invasiven.The photoprotein bolinopsin is suitable for the visualization of tissues or cells during surgical interventions, especially for invasive, non-invasive, and minimally invasive.
Das Photoprotein Bolinopsin eignet sich auch zur Markierung von Tumorgeweben und anderen phänotypisch veränderten Geweben speziell bei der histologischen Untersuchung, bei operativen Eingriffen.The photoprotein bolinopsin is also suitable for marking tumor tissues and other phenotypically modified tissues, especially for histological examinations and surgical interventions.
Die Erfindung betrifft auch die Reinigung des Photoprotein Bolinopsin speziell als wildtyp Protein, als Fusionsprotein, als mutagenisiertes Protein.The invention also relates to the purification of the photoprotein bolinopsin, especially as a wild-type protein, as a fusion protein, as a mutagenized protein.
Die Erfindung betrifft auch die Verwendung des Photoprotein Bolinopsin auf dem Gebiet der Kosmetik speziell von Badezusätzen, von Lotionen, von Seifen, von Körperfarben, von Zahncreme, von Körperpudern. (The invention also relates to the use of the photoprotein bolinopsin in the field of cosmetics, in particular bath additives, lotions, soaps, body colors, toothpaste, body powders. (
Die Erfindung betrifft auch die Verwendung des Photoprotein Bolinopsin zur Färbung speziell von Nahrungsmitteln, von Badezusätzen, von Tinte, von Textilien, von Kunststoffen.The invention also relates to the use of the photoprotein bolinopsin for coloring food, bath additives, ink, textiles and plastics.
Die Erfindung betrifft auch die Verwendung des Photoprotein Bolinopsin zur Färbung von Papier speziell von Grußkarten, von Papierprodukten, von Tapeten, von Bastelartikehi.The invention also relates to the use of the photoprotein bolinopsin for coloring paper, especially greeting cards, paper products, wallpapers, and handicrafts.
Die Erfindung betrifft auch die Verwendung des Photoprotein Bolinopsin zur Färbung von Flüssigkeiten speziell für Wasserpistolen, für Springbrunnen, für Getränke, für Eis.The invention also relates to the use of the photoprotein bolinopsin for coloring liquids, especially for water pistols, for fountains, for drinks, for ice.
Die Erfindung betrifft auch die Verwendung des Photoprotein Bolinopsin zur Herstellung von Spielwaren speziell von Fingerfarbe, von Schminke.The invention also relates to the use of the photoprotein bolinopsin for the production of toys, especially of finger paint, of make-up.
Die Erfindung betrifft Nukleinsäuremoleküle, die das Polypeptid offenbart durch SEQ LD NO: 2 kodieren.The invention relates to nucleic acid molecules which encode the polypeptide by SEQ LD NO: 2.
Die Erfindung betrifft das Polypeptid mit der Aminosauresequenz, die in SEQ LD NO: 2 offenbart ist. Die Erfindung bezieht sich des weiteren auf Nukleinsäuremoleküle, ausgewählt aus der Gruppe bestehend ausThe invention relates to the polypeptide with the amino acid sequence disclosed in SEQ LD NO: 2. The invention further relates to nucleic acid molecules selected from the group consisting of
a) Nukleinsäuremolekülen, die ein Polypeptid kodieren, welches die Aminosäuresequenz offenbart durch SEQ ID NO: 2 umfasst;a) nucleic acid molecules encoding a polypeptide which comprises the amino acid sequence disclosed by SEQ ID NO: 2;
b) Nukleinsäuremolekülen, welche die durch SEQ ID NO: 1 dargestellte Sequenz enthalten;b) nucleic acid molecules containing the sequence represented by SEQ ID NO: 1;
c) Nukleinsäuremolekülen, deren komplementärer Strang mit einem Nukleinsäuremolekül aus a) oder b) unter stringenten Bedingungen hybridisiert und welche die biologische Funktion eines Photoproteins aufweisen;c) nucleic acid molecules whose complementary strand hybridizes with a nucleic acid molecule from a) or b) under stringent conditions and which have the biological function of a photoprotein;
d) Nukleinsäuremolekülen, welche sich auf Grund der Degenerierung des genetischen Kodes von den unter c) genannten unterscheiden;d) nucleic acid molecules which differ from those mentioned under c) due to the degeneracy of the genetic code;
e) Nukleinsäuremolekülen, welche eine Sequenzhomologie von mindestens 95 % zu SEQ LD NO: 1 zeigen, und deren Proteinprodukt die biologische Funktion eines Photoproteins aufweist; unde) nucleic acid molecules which have a sequence homology of at least 95% to SEQ LD NO: 1 and whose protein product has the biological function of a photoprotein; and
f) Nukleinsäuremolekülen, welche eine Sequenzhomologie von mindestens 65 % zu SEQ ID NO: 1 zeigen, und deren Proteinprodukt die biologische Funktion eines Photoproteins aufweist.f) nucleic acid molecules which have a sequence homology of at least 65% to SEQ ID NO: 1 and whose protein product has the biological function of a photoprotein.
Die Erfindung betrifft auch Nukleinsäuremoleküle, die eine Sequenzhoπiologie von mindestens 95 %, 90 %, 85 %, 80 %, 75 %, 70 %, 65 % oder 60 % zu SEQ ID NO: 1 aufweisen und für ein Polypeptid kodieren, welches die Eigenschaften eines Photoproteins besitzt.The invention also relates to nucleic acid molecules which have a sequence hiiology of at least 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60% to SEQ ID NO: 1 and code for a polypeptide which has the properties of a photoprotein.
Die Erfindung betrifft die oben genannten Nukleinsäuremoleküle, bei denen die Sequenz einen funktionalen Promotor 5" zu der das Photoprotein kodierenden Sequenz enthält.The invention relates to the above-mentioned nucleic acid molecules in which the sequence contains a functional promoter 5 "to the sequence coding for the photoprotein.
Die Erfindung betrifft auch Nukleinsäuremoleküle wie vorhergehend beschrieben, die Bestandteil von rekombinanten DNA oder RNA Vektoren sind.The invention also relates to nucleic acid molecules as described above, which are part of recombinant DNA or RNA vectors.
Die Erfindung betrifft Organismen, die einen solchen Vektor enthalten.The invention relates to organisms which contain such a vector.
Die Erfindung bezieht sich auf Oligonukleotide mit mehr als 1O aufeinanderfolgenden Nukleotiden, die identisch oder komplementär zur DNA oder RNA Sequenz der Bolinopsin Moleküle oder der weiteren erfindungsgemäßen Molekülen sind.The invention relates to oligonucleotides with more than 10 consecutive nucleotides which are identical or complementary to the DNA or RNA sequence of the bolinopsin molecules or of the further molecules according to the invention.
Die Erfindung betrifft Photoproteine, die durch die vorhergehend beschriebenen Nukleotid- sequenzen kodiert sind. Die Erfindung bezieht sich auf Verfahren zur Expression der erfindungsgemäßen Photoprotein Polypeptide in Bakterien, eukaryontischen Zellen oder in in vitro Expressionssystemen.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.
Die Erfindung betrifft auch Verfahren zur Aufreinigung/Isolierung eines erfindungsgemäßen Photoprotein Polypeptides.The invention also relates to methods for purifying / isolating a photoprotein polypeptide according to the invention.
Die Erfindung bezieht sich auf Peptide mit mehr als 5 aufeinanderfolgenden Aminosäuren, die immunologisch durch Antikörper gegen die erfindungsgemäßen Photoproteme erkannt werden.The invention relates to peptides with more than 5 consecutive amino acids which are recognized immunologically by antibodies against the photoprotems according to the invention.
Die Erfindung betrifft die Verwendung der erfindungsgemäßen, für Photoproteine kodierende Nukleinsäuren als Marker- oder Reportergene, insbesondere für die pharmakologische Wirkstoffsuche und Diagnostik.The invention relates to the use of the nucleic acids according to the invention, coding for photoproteins, as marker or reporter genes, in particular for pharmacological search for active substances and diagnostics.
Die Erfindung betrifft die Verwendung der cifmdungsgemäßen Photoproteme bzw. eine erfindungsgemäße, für ein Photoprotein kodierende Nukleinsaüre als Marker oder Reporter bzw. als Marker- oder Reportergen.The invention relates to the use of the photoprotems according to the invention or to a nucleic acid according to the invention coding for a photoprotein as a marker or reporter or as a marker or reporter gene.
Die Erfindung betrifft die Verwendung des Photoproteins Bolinopsin. (SEQ JJD NO: 2) bzw. die Verwendung einer für das Photoprotein Bolinopsin kodierenden Nvxideinsäure als Marker oder Reporter bzw. als Marker oder Reportergen insbesondere für die pharmakologische Wirkstoffsuche und Diagnostik.The invention relates to the use of the photoprotein bolinopsin. (SEQ JJD NO: 2) or the use of an Nvxideinsäure coding for the photoprotein bolinopsin as a marker or reporter or as a marker or reporter gene, in particular for pharmacological drug discovery and diagnostics.
Die Erfindung betrifft die Verwendung der in SEQ ID NO: 1 dargestellten Nukleinsaüre als Marker- oder Reportergen, insbesondere für die pharmakologische Wirikstoffsuche und Diagnostik.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 pharmacological drug search and diagnostics.
Gegenstand der Erfindung sind auch polyklonale oder monoklonale Avntikörper, welche ein erfin- dungsgemäßes Polypeptid erkennen.The invention also relates to polyclonal or monoclonal antibodies which recognize a polypeptide according to the invention.
Die Erfindung betrifft auch monoklonale oder polyklonale Antikörper, die das Photoprotein Bolinopsin (SEQ ID NO: 2) erkennen.The invention also relates to monoclonal or polyclonal antibodies which recognize the photoprotein bolinopsin (SEQ ID NO: 2).
Expression der erfindungsgemäßen PhotoproteineExpression of the photoproteins according to the invention
Als Expression bezeichnet man die Produktion eines Moleküls, das nach dem Einbringen des Gens in eine geeignete Wirtszelle die Transcription und Translation des in einen Expressionsvektor Monierte Fremdgen erlaubt. Expressionsvektoren enthalten die für die Expression von Genen in Zellen von Prokaryoten oder Eukaryonten erforderlichen Kontrollsignale.Expression refers to the production of a molecule which, after the gene has been introduced into a suitable host cell, allows 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.
Expressionsvektoren können prinzipiell auf zwei verschiedene Weisen konstruiert werden. Bei den sogenannten Transcriptionsfusionen wird das vom einklonierten Fremdgen codierte Protein als authentisches, biologisch aktives Protein synthetisiert. Der Expressionsvektor trägt hierzu alle zur Expression benötigten 5 - und 3 - Kontrollsignale.In principle, expression vectors can be constructed in two different ways. In so-called transcription fusions, the protein encoded by the cloned-in foreign gene is called authentic, biologically active protein synthesized. For this purpose, the expression vector carries all 5 and 3 control signals required for expression.
Bei den sogenannten Translationsfusionen wird das vom einklonierten Fremdgen codierte Protein als Hybridprotein zusammen mit einem anderen Protein exprimiert, das sich leicht nachweisen lässt. Die zur Expression benötigten 5 - und 3V- Kontrollsignale inklusive des Startcodons und eventuell ein Teil der für die N-terminalen Bereiche des zu bildenden Fϊybridproteins codierenden Sequenzen stammen vom Vektor. Der zusätzliche eingeführte Proteinteil stabilisiert nicht nur in vielen Fällen das vom einklonierten Fremdgen codierte Protein vor dem Abbau durch zelluläre Proteasen, sondern lässt sich auch zum Nachweis und zur Isolierung des gebildeten Hybridproteins einsetzen. Die Expression kann sowohl transient, als auch stabil erfolgen. Als Wirtsorganismen eignen sich sowohl Bakterien, Hefen, Viren als auch eukaryotische Systeme.In so-called translation fusions, the protein encoded by the cloned-in foreign gene is expressed as a hybrid protein together with another protein that can be easily detected. The 5 and 3 V control signals required for expression, including the start codon and possibly some of the sequences coding for the N-terminal regions of the hybrid protein to be formed, come from the vector. The additional protein part introduced not only stabilizes the protein encoded by the cloned-in foreign gene in many cases before it is broken down by cellular proteases, but can also be used for the detection and isolation of the hybrid protein formed. Expression can be both transient and stable. Both bacteria, yeasts, viruses and eukaryotic systems are suitable as host organisms.
Reinigung der erfindungsgemäßen PhotoproteinePurification of the photoproteins according to the invention
Die Isolierung von Proteinen (auch nach Überexpression) wird häufig als Proteinreinigung bezeichnet. Zur Proteinreinigung steht eine Vielzahl an etablierten Methoden und Verfahren zur Verfügung.The isolation of proteins (even after overexpression) is often referred to as protein purification. A variety of established methods and processes are available for protein purification.
Die Fest-Flüssig-Trennung ist eine Grundoperation bei Proteinisolierungen. Sowohl bei der Abtrennung der Zellen vom Kulturmedium als auch bei der Klärung des Rohextraktes nach Zellaufschluss und Entfernung der Zelltrümmer, bei der Abtrennung von Niederschlägen nach Fällungen usw. ist der Verfahrensschritt erforderlich. Er erfolgt durch Zentrifugation und Filtration.Solid-liquid separation is a basic operation in protein isolation. The process step is required 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 the cell debris, in the separation of precipitates after precipitation, etc. It is done by centrifugation and filtration.
Durch Gewinnung intrazellulärer Proteine muss die Zellwand zerstört bzw. durchlässig gemacht werden. Je nach Maßstab und Organismus werden dazu Hochdruckhomogenisatoren oder Rührwerkskugel- bzw. Glasperlenmühlen eingesetzt. Im Labormaßstab kommen u.a. mechanische Zellintegrationen und Ultraschallbehandlung zum Einsatz.The cell wall must be destroyed or made permeable by obtaining intracellular proteins. Depending on the scale and organism, high-pressure homogenizers or agitator ball or glass bead mills are used. On a laboratory scale, mechanical cell integrations and ultrasound treatment are used.
Sowohl für extrazelluläre als auch intrazelluläre Proteine (nach Zellaufschluss) sind verschiedene Fällungsverfahren mit Salzen (insbesondere Ammoniumsulfat) oder organischen Lösungsmitteln (Alkohole, Aceton) eine schnelle und effiziente Methode zur Konzentration von Proteinen. Bei der Reinigung intrazellulärer Proteine ist die Entfernung der löslichen Nukleinsäuren erstrebenswert (Fällung z.B. mit Streptomycin- oder Protaminsulfat). Bei der Gewinnung extrazellulärer Proteine werden häufig Träger (z.B. Stärke, Kieselgur) vor Zugabe der Fällungsmittel zugesetzt, um besser handhabbare Niederschläge zu erhalten. Für die Feinreinigung stehen zahlreiche chromatographische und Verteilungsverfahren zur Verfügung (Absorptions- und Ionenaustauschchromatographie, Gelfiltration, Affinitätschromatographie, Elektrophoresen). Eine Säulenchromatographie wird auch im technischen Maßstab angewandt. Für den Labormaßstab ist vor allem die Affinitätschromatographie von Bedeutung, die Reinigungsfaktoren bis zu mehreren 100 pro Schritt ermöglicht.For both extracellular and intracellular proteins (after cell disruption), various precipitation processes with salts (in particular ammonium sulfate) or organic solvents (alcohols, acetone) are a quick and efficient method of concentrating proteins. In the purification of intracellular proteins, the removal of the soluble nucleic acids is desirable (precipitation, for example with streptomycin or protamine sulfate). When extracellular proteins are obtained, carriers (eg starch, diatomaceous earth) are often added before the precipitants are added in order to obtain more manageable precipitates. Numerous chromatographic and distribution processes are available for fine purification (absorption and ion exchange chromatography, gel filtration, affinity chromatography, electrophoresis). Column chromatography is also used on an industrial scale. Affinity chromatography, which enables purification factors of up to several hundred per step, is of particular importance for the laboratory scale.
Extrazelluläre Proteine fallen in relativ verdünnten Lösungen an. Sie müssen ebenso wie extrazelluläre Proteine vor ihrer weiteren Verwendung konzentriert werden. Neben den schon erwähnten Verfahren hat sich - auch im industriellen Maßstab - die Ultrafiltration bewährt.Extracellular proteins are obtained in relatively dilute solutions. Like extracellular proteins, they must be concentrated before further use. In addition to the processes already mentioned, ultrafiltration has also proven itself - also on an industrial scale.
Anorganische Salze als Begleitstoffe von Proteinen sind für spezifische Anwendungen häufig unerwünscht. Sie können u.a. durch Gelfiltration, Dialyse und Diafiltration entfernt werden.Inorganic salts as accompanying substances in proteins are often undesirable for specific applications. Among other things, you can removed by gel filtration, dialysis and diafiltration.
Zahlreiche Proteine kommen als Trockenpräparate zum Einsatz. Als Trocknungsverfahren sind die Vakuum-, Gefrier- und Sprühtrocknung von Bedeutung.Numerous proteins are used as dry preparations. Vacuum, freeze and spray drying are important drying methods.
Nukleotid- und AminosäuresequenzenNucleotide and amino acid sequences
Das Photoprotein Bolinopsin wird durch die folgende Nukleotidsequenz kodiert (SEQ ID NO: 1):The photoprotein bolinopsin is encoded by the following nucleotide sequence (SEQ ID NO: 1):
5 -5 -
ATGCCTCTAGACGAGACCAACAACGAAAGCTATAGATGGCTGAGAAGTGTGGGTAAC GATTGGCAGTTTGATGTCGAGGACGTTCATCCTAAACAGCTTAGTCGGCTCTACAAGA GATTCGACACCTTCGATCTAGACAGTGACGGTCGTATGGACATGGACGAGATCCTGTA CTGGCCCGACAGAATGAGGCAGCTGGTGAACGCTTCTGACGAACAGGTCGAGAAGAT GAGGGCTGCTTGCTACACCTTCTTCCACAACAAAGGAGTGGATCCAGAAAAGGGACTC CTCAGAGACGACTGGGTTGAGGCTAACAGAGTATTTGCTGAGGCTGAAAGAGAGAGG GAACGACGTGGCATGCCCTCCTTGATTGGTCTTTTGTCAGACGCTTACTACGATGTCCT GGATGATGACGGTGATGGTACTGTTGATGTTGATGAACTCAAAACCATGATGAAGGCT TTTGATGTGCCCCAGGAGGCTGCCTACACCTTCTTTAAGAAA.GCTGACACGGATAATA GTGGAAAACTGGAGAGAAGCGAACTGGTCCATCTCTTCAGAAAGTTCTGGATGGAATC CTACGATCCTCAGTGGGACGGTGTCTACGCTTACAAATATTA-A-3\ATGCCTCTAGACGAGACCAACAACGAAAGCTATAGATGGCTGAGAAGTGTGGGTAAC GATTGGCAGTTTGATGTCGAGGACGTTCATCCTAAACAGCTTAGTCGGCTCTACAAGA GATTCGACACCTTCGATCTAGACAGTGACGGTCGTATGGACATGGACGAGATCCTGTA CTGGCCCGACAGAATGAGGCAGCTGGTGAACGCTTCTGACGAACAGGTCGAGAAGAT GAGGGCTGCTTGCTACACCTTCTTCCACAACAAAGGAGTGGATCCAGAAAAGGGACTC CTCAGAGACGACTGGGTTGAGGCTAACAGAGTATTTGCTGAGGCTGAAAGAGAGAGG GAACGACGTGGCATGCCCTCCTTGATTGGTCTTTTGTCAGACGCTTACTACGATGTCCT GGATGATGACGGTGATGGTACTGTTGATGTTGATGAACTCAAAACCATGATGAAGGCT TTTGATGTGCCCCAGGAGGCTGCCTACACCTTCTTTAAGAAA.GCTGACACGGATAATA GTGGAAAACTGGAGAGAAGCGAACTGGTCCATCTCTTCAGAAAGTTCTGGATGGAATC CTACGATCCTCAGTGGGACGGTGTCTACGCTTACAAATATTA-A-3 \
Daraus ergibt sich eine Aminosäuresequenz von (SEQ ID NO: 2):This results in an amino acid sequence of (SEQ ID NO: 2):
MPLDETNNESYRWLRSVGNDWQFDVEDVHPKQLSP^YΈSFTJΓFDLDSDGRMDMDEΓLY WPDRMRQLVNASDEQVEKMRAACYTFFHNKGVDPEKGLLRDX)WVEANRVFAEAERERE RRGMPSLIGLLSDAYYDVLDDDGDGTVDVDELKTMMKAFDVPQEAAYTFFKKADTDNS GKLERSELVHLFRKFWMESYDPQWDGVYAYKY Diese Sequenzen finden sich im Sequenzlisting wieder.MPLDETNNESYRWLRSVGNDWQFDVEDVHPKQLSP ^ YΈSFTJΓFDLDSDGRMDMDEΓLY WPDRMRQLVNASDEQVEKMRAACYTFFHNKGVDPEKGLLRDX) WVEANRVFAEAERERE RRGMPSLIGLLSDAYYDVLDDDGDGTVDVDELKTMMKAFDVPQEAAYTFFKKADTDNS GKLERSELVHLFRKFWMESYDPQWDGVYAYKY These sequences can be found in the sequence listing.
Kurze Beschreibung der FigurenBrief description of the figures
Fig. 1: Die Fig. 1 zeigt die Plasmidkarte des Vektors pTriplEX2-Bolinopsin.Fig. 1: Fig. 1 shows the plasmid map of the vector pTriplEX2-bolinopsin.
Fig. 2: Die Fig. 2 zeigt die Plasmidkarte des Vektors pcDNT A3 -BolinopsinFig. 2: Fig. 2 shows the plasmid map of the vector pcDNT A3 -Bolinopsin
Fig. 3: Die Fig. 3 zeigt die Exitation von Bolinopsin. Y : Intensität; X : Wellenlänge [um].Fig. 3: Fig. 3 shows the exitation of bolinopsin. Y: intensity; X: wavelength [um].
Fig. 4: Die Fig. 4 zeigt die Fluoreszenz von Bolinopsin. Y : Intensität; X : Wellenlänge [nm] .Fig. 4: Fig. 4 shows the fluorescence of bolinopsin. Y: intensity; X: wavelength [nm].
Fig. 5: Die Fig. 5 zeigt die Biolumineszenz von Bolinopsin. Y : Intensität; X : Wellenlänge [nm].Fig. 5: Fig. 5 shows the bioluminescence of bolinopsin. Y: intensity; X: wavelength [nm].
Fig. 6: Die Fig. 6 zeigt das Alignment von Bolinopsin und Aequorin (aequoria victoria) auf Nukleinsäureebene.Fig. 6: Fig. 6 shows the alignment of bolinopsin and aequorin (aequoria victoria) at the nucleic acid level.
Fig. 7: Die Fig. 7 zeigt das Alignment von Bolinopsin und Aequorin (aequoria victoria) auf Aminosäureebene.Fig. 7: Fig. 7 shows the alignment of bolinopsin and aequorin (aequoria victoria) at the amino acid level.
Fig. 8: Die Fig. 8 zeigt das Ergebnis der Biolumineszenzmessung von Bolinopsin nach bakterieller Expression. Y : Lumineszenz in RLU [relative light units]; X : μl Lysat : 0 = uninduziertes Kontrolllysat.Fig. 8: Fig. 8 shows the result of the bioluminescence measurement of bolinopsin after bacterial expression. Y: luminescence in RLU [relative light units]; X: μl lysate: 0 = uninduced control lysate.
Fig. 9: Die Fig. 9 zeigt das Ergebnis der Biolumineszenzmessung von Bolinopsin nach bakterieller Expression in Abhängigkeit vom eingesetzten Coelenterazinderivat. Y : Lumineszenz in RLU [relative light units]; X : Coelenterazinderivat : 1 = nativ, 2 = cp, 3 = f , 4 = fcp, 5 = hcp, 6 = h, 7 = i, 8 = ip, 9 = n; Balken : schwarz : uninduziertes Kontrolllysat; hell-grau : 10 μl Lysat; weiss : 20 μl Lysat; dunkel-grau : 40 μl Lysat. FIG. 9: FIG. 9 shows the result of the bioluminescence measurement of bolinopsin after bacterial expression as a function of the coelenterazine derivative used. Y: luminescence in RLU [relative light units]; X: Coelenterazine derivative: 1 = native, 2 = cp, 3 = f, 4 = fcp, 5 = hcp, 6 = h, 7 = i, 8 = ip, 9 = n; Bars: black: uninduced control lysate; light gray: 10 μl lysate; white: 20 μl lysate; dark gray: 40 μl lysate.
BeispieleExamples
Beispiel 1example 1
Als Vektor zur Herstellung des im folgenden dargestellten Konstruktes wurde das Plasmid pTriplEx2 der Firma Clontech verwendet. Das Derivat des Vektors wurde als pTriplEx2- 5 Bolinopsin bezeichnet. Der Vektor pTriplEx2-Bolinopsin wurde zur Expression von Bolinospin in bakteriellen Systemen verwendet.The plasmid pTriplEx2 from Clontech was used as the vector for producing the construct shown below. The derivative of the vector was named pTriplEx2- 5 bolinopsin. The vector pTriplEx2-bolinopsin was used to express bolinospin in bacterial systems.
Die Fig. 1 zeigt die Plasmidkarte des Vektors pTriplEX2-Bolinopsin .1 shows the plasmid map of the vector pTriplEX2-bolinopsin.
Beispiel 2Example 2
Als Vektor . zur Herstellung des im folgenden dargestellten Konstruktes wurde das PlasmidO pcDNA3.1(+) der Firma Clontech verwendet. Das Derivat des Vektors wurde als pcDNA3- Bolinopsin bezeichnet. Der Vektor pcDNA3-Bolinopsin wurde zur Expression von Bolinospin in eukaryotischen Systemen verwendet.As a vector. the plasmidO pcDNA3.1 (+) from Clontech was used to produce the construct shown below. The derivative of the vector was named pcDNA3-bolinopsin. The vector pcDNA3-bolinopsin was used to express bolinospin in eukaryotic systems.
Die Fig. 2 zeigt die Plasmidkarte des Vektors pcDl A3-Bolinopsm .2 shows the plasmid map of the vector pcDl A3-Bolinopsm.
Beispiel 3 5 Bakterielle ExpressionExample 3 5 Bacterial Expression
Die bakterielle Expression erfolgte im E. coli Stamm BL21(DE3). durch Transformation der Bakterien mit den Expressionsplasmiden pTriplEX2-Bolinopsin und pTriplEX2. Die transformierten Bakterien wurden in LB-Medium bei 37°C für 3 Stunden inkubiert und die Expression für 4 Stunden durch Zugabe von IPTG bis zu einer Endkonzentration von 1 mM induziert. DieO induzierten Bakterien wurden durch Zentrifugation geerntet, in PBS + 5 mM EDTA resuspendiert und durch Ultraschall aufgeschlossen. Das Lysat wurde 3 Stunden mit Coelenterazin im dunkeln inkubiert. Direkt nach der Zugabe von 5 mM Calziumchlόrid wurde die Biolumineszenz im Luminometer gemessen. Die Integrationszeit der Messung betrug 40 Sekunden.Bacterial expression took place in the E. coli strain BL21 (DE3). by transforming the bacteria with the expression plasmids pTriplEX2-bolinopsin and pTriplEX2. The transformed bacteria were incubated in LB medium at 37 ° C. for 3 hours and expression was induced for 4 hours by adding IPTG to a final concentration of 1 mM. The O-induced bacteria were harvested by centrifugation, resuspended in PBS + 5 mM EDTA and disrupted by ultrasound. The lysate was incubated with coelenterazine in the dark for 3 hours. Immediately after the addition of 5 mM calcium chloride, the bioluminescence was measured in the luminometer. The integration time of the measurement was 40 seconds.
Die Fig. 8 zeigt die Ergebnisse der Biolumineszenzmessung von Bolinopsin. 5 Beispiel 48 shows the results of the bioluminescence measurement of bolinopsin. 5 Example 4
Eukaryotische ExpressionEukaryotic expression
Die konstitutive eukaryotische Expression erfolgte in CHO-Zellen durch Transfektion der Zellen mit den Expressionsplasmiden pcDNA3-Bolinopsin und pcDNA3.1(+) in transienten Experi- menten. Hierzu wurden 10000 Zellen pro Loch in DMEM-F12 Medium auf 96 Loch ikrotiter- platten plattiert und über Nacht bei 37°C inkubiert. Die Transfektion erfolgte mit Hilfe des Fugene 6 Kits (Röche) nach Herstellerangaben. Die transfizierten Zellen wurden über Nacht bei 37°C in DMEM-F12 Medium inkubiert.The constitutive eukaryotic expression was carried out in CHO cells by transfection of the cells with the expression plasmids pcDNA3-bolinopsin and pcDNA3.1 (+) in transient experiments. instruments. For this purpose, 10,000 cells per well were plated in DMEM-F12 medium on 96 well microtiter plates and incubated at 37 ° C. overnight. The transfection was carried out using the Fugene 6 kit (Röche) according to the manufacturer's instructions. The transfected cells were incubated overnight at 37 ° C in DMEM-F12 medium.
Beispiel 5Example 5
BLASTBLAST
Ergebnis einer BLAST-Analyse von Bolinopsin auf der Aminosäureebene.Result of a BLAST analysis of bolinopsin at the amino acid level.
>pdb|lJF2|A Chain A, Crystal Structure Of W92f Obelin Mutant From Obelia Longissima At 1.72 Angstrom Resolution, Length = 195, Score = 85.1 bits (209), Expect = 8e-16, Identities = 52/177 (29%), Positives = 90/177 (50%), Gaps = 4/177 (2%)> pdb | lJF2 | A Chain A, Crystal Structure Of W92f Obelin Mutant From Obelia Longissima At 1.72 Angstrom Resolution, Length = 195, Score = 85.1 bits (209), Expect = 8e-16, Identities = 52/177 (29%) , Positives = 90/177 (50%), gaps = 4/177 (2%)
>emb|CAD87698.1| unnamed protein product [synthetic construct], Length = 195, Score => Emb | CAD87698.1 | unnamed protein product [synthetic construct], Length = 195, Score =
81.6 bits (200), Expect = 8e-15, Identities = 51/177 (28%), Positives = 89/177 (499b), Gaps = 4/177 (2%)81.6 bits (200), expect = 8e-15, identities = 51/177 (28%), positives = 89/177 (499b), gaps = 4/177 (2%)
>pdb|lJF0|A Chain A, The Crystal Structure Of Obelin From Obelia Geniculata At 1.82 A Resolution, gb|AAL86372.1|AF394688_l apoobelin [Obelia geniculata], Length = 195, Score = 80.1 bits (196), Expect = 2e-14, Identities = 51/177 (28%), Positives = 89/177 (49%), Gaps = 4/177 (2%)> pdb | lJF0 | A Chain A, The Crystal Structure Of Obelin From Obelia Geniculata At 1.82 A Resolution, gb | AAL86372.1 | AF394688_l apoobelin [Obelia geniculata], Length = 195, Score = 80.1 bits (196), Expect = 2e -14, Identities = 51/177 (28%), Positive = 89/177 (49%), Gaps = 4/177 (2%)
>sp|P39047|MYTR_MITCE Mitrocomin precursor, pir||S39022 mitrocomin precursor - rv itrocoma cellularia, gb|AAA29298.1| apomitrocornin, Length = 198, Score = 78.6 bits (192), Expect = 7e-14, Identities = 47/177 (26%), Positives = 91/177 (50%), Gaps = 4/177 (2%)> sp | P39047 | MYTR_MITCE Mitrocomin precursor, pir || S39022 mitrocomin precursor - rv itrocoma cellularia, gb | AAA29298.1 | apomitrocornin, Length = 198, Score = 78.6 bits (192), Expect = 7e-14, Identities = 47/177 (26%), Positive = 91/177 (50%), Gaps = 4/177 (2%)
>sp|Q08121|CLYT_CLYGR Clytin precursor (Phialidin), pir||S28860 clytin - hydromedusa (Clytia gregarium), emb|CAA49754.1| clytin [Clytia gregaria], gb|AAA28293.1| apoclytin, Length = 198, Score = 77.4 bits (189), Expect = 2e-13, Identities = 53/177 (29%), Positives = 89/177 (49%), Gaps = 4/177 (2%)> sp | Q08121 | CLYT_CLYGR Clytin precursor (phialidine), pir || S28860 clytin - hydromedusa (Clytia gregarium), emb | CAA49754.1 | clytin [Clytia gregaria], gb | AAA28293.1 | apoclytin, length = 198, score = 77.4 bits (189), expect = 2e-13, identities = 53/177 (29%), positives = 89/177 (49%), gaps = 4/177 (2%)
Beispiel 6Example 6
BLASTBLAST
Ergebnis einer BLAST-Analyse von Bolinopsin auf Nukleinsäureebene.Result of a BLAST analysis of bolinopsin at the nucleic acid level.
>gb|AC073341.10| Homo sapiens BAC clone RP11-549I23 from 7, complete sequence, Length = 185574, Score = 52.6 bits (27), Expect = 4e-04, Identities = 33/36 (91%) >gb|AC092850.13| Homo sapiens 12 BAC RP11-346B9 (Roswell Park Cancer Institute Human BAC Library) complete sequence, Length = 176733, Score = 46.8 bits (24), Expect = 0.023, Identities = 32/36 (88%)> Gb | AC073341.10 | Homo sapiens BAC clone RP11-549I23 from 7, complete sequence, Length = 185574, Score = 52.6 bits (27), Expect = 4e-04, Identities = 33/36 (91%) > Gb | AC092850.13 | Homo sapiens 12 BAC RP11-346B9 (Roswell Park Cancer Institute Human BAC Library) complete sequence, Length = 176733, Score = 46.8 bits (24), Expect = 0.023, Identities = 32/36 (88%)
>gb|AC126564.7| Homo sapiens 12 BAC RP11-638F5 (Roswell Park Cancer Institute Human BAC Library) complete sequence, Length = 121242, Score = 46.8 bits (24), Expect = 0.023, Identities = 32/36 (88%)> Gb | AC126564.7 | Homo sapiens 12 BAC RP11-638F5 (Roswell Park Cancer Institute Human BAC Library) complete sequence, Length = 121242, Score = 46.8 bits (24), Expect = 0.023, Identities = 32/36 (88%)
>gb|AC093924.3| Genomic sequence for Mus musculus, clone RP23-239M9, from chromosome 17, complete sequence, Length = 166277, Score = 44.9 bits (23), Expect = 0.086, Identities = 31/35 (88%)> Gb | AC093924.3 | Genomic sequence for Mus musculus, clone RP23-239M9, from chromosome 17, complete sequence, Length = 166277, Score = 44.9 bits (23), Expect = 0.086, Identities = 31/35 (88%)
>gb|AC060234.11| Homo sapiens chromosome 10 clone RP11-523O18, complete sequence, Length = 170073, Score = 44.9 bits (23), Expect = 0.O86, Identities = 31/35 (88%)> Gb | AC060234.11 | Homo sapiens chromosome 10 clone RP11-523O18, complete sequence, Length = 170073, Score = 44.9 bits (23), Expect = 0.O86, Identities = 31/35 (88%)
>gb|AC084727.14| Homo sapiens chromosome 10 clone RP11-507P23, complete sequence, Length = 188652, Score = 44.9 bits (23), Expect = 0.086, Identities = 31/35 (88%)> Gb | AC084727.14 | Homo sapiens chromosome 10 clone RP11-507P23, complete sequence, Length = 188652, Score = 44.9 bits (23), Expect = 0.086, Identities = 31/35 (88%)
Beispiel 7Example 7
Die Fig. 6 zeigt das Alignment von Bolinopsin mit Aequorin (Aequoria victoria) auf Nukleinsäureebene.6 shows the alignment of bolinopsin with aequorin (Aequoria victoria) at the nucleic acid level.
Beispiel 8Example 8
Die Fig. 7 zeigt das Alignment von Bolinopsin mit Aequorin (Aequoria victoria) auf Aminosäureebene.7 shows the alignment of bolinopsin with aequorin (Aequoria victoria) at the amino acid level.
Beispiel 9Example 9
Spektrum des Photoproteins BolinopsinSpectrum of the photoprotein bolinopsin
Zur Messung der spektralen Eigenschaften von Bolinopsin wurden E. coli BL21(DE3) mit den Plasmiden pTriplEX2-CGFP und pTriplEX2 transformiert. Die Induktion erfolgte durch die Zugabe von 1 rnM IPTG und einer Inkubation von 4 Stunden bei 37°C. Anschließend wurden die Bakterien geerntet und in PBS resuspendiert. Die Lyse erfolgte durch Ultraschall. Anschließend erfolgte die Messung der Fluoreszenz bzw. Biolumineszenz. Das Maximum der Exitation lag bei 352 nm, der Fluoreszenz bei 452 nm und das der Biolumineszenz bei 468 nm.To measure the spectral properties of bolinopsin, E. coli BL21 (DE3) were transformed with the plasmids pTriplEX2-CGFP and pTriplEX2. The induction was carried out by adding 1 nmM IPTG and incubating for 4 hours at 37 ° C. The bacteria were then harvested and resuspended in PBS. The lysis was carried out by ultrasound. The fluorescence or bioluminescence was then measured. The maximum of the excitation was 352 nm, the fluorescence at 452 nm and that of the bioluminescence at 468 nm.
Die Fig. 3 zeigt die Exitation von Bolinopsin Die Fig. 4 zeigt die Fluoreszenz von Bolinopsin Die Fig. 5 zeigt die Biolumineszenz von Bolinopsin3 shows the exitation of bolinopsin. FIG. 4 shows the fluorescence of bolinopsin 5 shows the bioluminescence of bolinopsin
Beispiel 10Example 10
Zur Identifizierung von Substraten für Bolinopsin wurden 5 μl Lösungen verschiedener Coelenterazinderivate (10"4 M) in Methanol mit jeweils 0, 10, 20 und 40 μl Lysat in einem Gesamtvolumen von 75 μl für 3 Stunden bei 4°C inkubiert und die Lumineszenz nach der Zugabe von 25 μl Calziumchlorid (Endkonzentration 5 mM) gemessen. Die Coelenterazine wurden von Sigma (Deutschland) bezogen. Bolinopsin zeigte mit allen eingesezten Coelenterazinderivaten Biolumineszenzaktivität. Die höchste Aktivität könnte mit dem nativen Coelenterazin gemessen werden.To identify substrates for bolinopsin, 5 ul solutions of various coelenterazine derivatives (10 "4 M) in methanol with 0, 10, 20 and 40 ul lysate in a total volume of 75 ul were incubated for 3 hours at 4 ° C and the luminescence after Addition of 25 μl calcium chloride (final concentration 5 mM) measured. The coelenterazines were obtained from Sigma (Germany). Bolinopsin showed bioluminescence activity with all coelenterazine derivatives used. The highest activity could be measured with the native coelenterazine.
Die Fig. 9 zeigt die Coelenterazin-Derivate als potentielle Substrate für Bolinopsin und eine grafische Darstellung der Lumineszenzmessung für 30 Sekunden bei 8,7 kV im Luminometer (RLU, relative light units).FIG. 9 shows the coelenterazine derivatives as potential substrates for bolinopsin and a graphic representation of the luminescence measurement for 30 seconds at 8.7 kV in the luminometer (RLU, relative light units).
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Claims

Patentansprüche claims
1. Nukleinsäuremolekül, ausgewählt aus der Gruppe bestehend aus a) Nukleinsäuremolekülen, die ein Polypeptid kodieren, welches die Aminosäuresequenz offenbart durch SEQ ID NO: 2 umfasst; b) Nukleinsäuremolekülen, welche die in SEQ ID NO: 1 dargestellte Sequenz umfassen; c) Nukleinsäuremolekülen, deren komplementärer Strang mit einem Nukleinsäuremolekül aus a) oder b) unter stringenten Bedingungen hybridisiert und welche die biologische Funktion eines Photoproteins aufweisen; d) Nukleinsäuremolekülen, welche sich auf Grund der Degenerierung des genetischen Kodes von den unter c) genannten unterscheiden; e) Nukleinsäuremolekülen, welche eine Sequenzhomologie von mindestens 95 % zu SEQ ID NO: 1 zeigen, und welche die biologische Funktion eines Photoproteins aufweisen; und f) Nukleinsäuremolekülen, welche eine Sequenzhomologie von mindestens 65% zu SEQ ID NO: 1 zeigen, und welche die biologische Funktion eines Photoproteins aufweisen.1. nucleic acid molecule selected from the group consisting of a) nucleic acid molecules which encode a polypeptide which comprises the amino acid sequence disclosed by SEQ ID NO: 2; b) nucleic acid molecules comprising the sequence shown in SEQ ID NO: 1; c) nucleic acid molecules whose complementary strand hybridizes with a nucleic acid molecule from a) or b) under stringent conditions and which have the biological function of a photoprotein; d) nucleic acid molecules which differ from those mentioned under c) due to the degeneracy of the genetic code; e) nucleic acid molecules which show a sequence homology of at least 95% to SEQ ID NO: 1 and which have the biological function of a photoprotein; and f) nucleic acid molecules which show a sequence homology of at least 65% to SEQ ID NO: 1 and which have the biological function of a photoprotein.
2. Nukleinsaüre nach Anspruch 1 , welche einen funktionalen Promotor 5^ zur das Photoprotein kodierenden Sequenz enthält.2. Nucleic acid according to claim 1, which contains a functional promoter 5 ^ to the sequence coding for the photoprotein.
3. Rekombinante DNA oder RNA Vektoren, welche Nukleinsäuren nach Anspruch 2 enthalten.3. Recombinant DNA or RNA vectors which contain nucleic acids according to claim 2.
4. Organismen, die einen Vektor gemäß Anspruch 3 enthalten.4. Organisms containing a vector according to claim 3.
5. Oligonukleotide mit mehr als 10 aufeinanderfolgenden Nukleotiden, die identisch, oder komplementär zu einer Teilsequenz eines Nukleinsäuremoleküls gemäß Anspruch 1 sind.5. Oligonucleotides with more than 10 consecutive nucleotides which are identical or complementary to a partial sequence of a nucleic acid molecule according to claim 1.
6. Polypeptid, das durch eine Nukleinsäuresequenz nach Anspruch 1 kodiert ist.6. polypeptide which is encoded by a nucleic acid sequence according to claim 1.
7. Verfahren zur Expression der Photoprotein Polypeptide gemäss Anspruch 6 in Bakterien, eukaryontischen Zellen oder in in vitro Expressionssystemen. 7. A method for expressing the photoprotein polypeptides according to claim 6 in bacteria, eukaryotic cells or in in vitro expression systems.
8. Verfahren zur Aufreinigung/Isolierung eines Photoprotein Polypeptides gemäss Anspruch 6.8. A method for purifying / isolating a photoprotein polypeptide according to claim 6.
9. Peptide mit mehr als 5 aufeinanderfolgenden Aminosäuren, die immunologisch durch Antikörper gegen das Photoprotein Bolinopsin erkannt werden.9. Peptides with more than 5 consecutive amino acids that are recognized immunologically by antibodies against the photoprotein bolinopsin.
10. Verwendung einer für ein Photoprotein kodierende Nukleinsaüre gemäß den Ansprüchen 1 bis 3 als Marker- oder Reportergen.10. Use of a nucleic acid coding for a photoprotein according to claims 1 to 3 as a marker or reporter gene.
11. Verwendung eines Photoproteins gemäß Anspruch 6 als Marker oder Reporter. 11. Use of a photoprotein according to claim 6 as a marker or reporter.
EP04740054A 2003-06-23 2004-06-18 Isolated photoprotein bolinopsin, and the use thereof Withdrawn EP1638998A1 (en)

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Title
INOUYE S. ET AL.: "Cloning and sequence analysis of cDNA for the Ca2+-activated photoprotein, clytin", FEBS LETTERS, vol. 315, no. 3, January 1993 (1993-01-01), pages 343 - 346, XP025593147, DOI: doi:10.1016/0014-5793(93)81191-2 *
See also references of WO2005000885A1 *

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