WO2005000885A1 - Photoproteine isolee « bolinopsine », et son utilisation - Google Patents

Photoproteine isolee « bolinopsine », et son utilisation Download PDF

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Publication number
WO2005000885A1
WO2005000885A1 PCT/EP2004/006608 EP2004006608W WO2005000885A1 WO 2005000885 A1 WO2005000885 A1 WO 2005000885A1 EP 2004006608 W EP2004006608 W EP 2004006608W WO 2005000885 A1 WO2005000885 A1 WO 2005000885A1
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WO
WIPO (PCT)
Prior art keywords
photoprotein
nucleic acid
bolinopsin
sequence
acid molecules
Prior art date
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PCT/EP2004/006608
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German (de)
English (en)
Inventor
Stefan Golz
Svetlana Markova
Ludmila Burakova
Ludmila Frank
Eugene Vysotski
Original Assignee
Bayer Healthcare Ag
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Filing date
Publication date
Application filed by Bayer Healthcare Ag filed Critical Bayer Healthcare Ag
Priority to EP04740054A priority Critical patent/EP1638998A1/fr
Publication of WO2005000885A1 publication Critical patent/WO2005000885A1/fr

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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

L'invention concerne la photoprotéine bolinopsine, sa séquence nucléotidique et sa séquence d'acides aminés, ainsi que son activité et son utilisation.
PCT/EP2004/006608 2003-06-23 2004-06-18 Photoproteine isolee « bolinopsine », et son utilisation WO2005000885A1 (fr)

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Application Number Priority Date Filing Date Title
EP04740054A EP1638998A1 (fr) 2003-06-23 2004-06-18 Photoproteine isolee bolinopsine , et son utilisation

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Application Number Priority Date Filing Date Title
DE10328067.7 2003-06-23
DE2003128067 DE10328067A1 (de) 2003-06-23 2003-06-23 Isoliertes Photoprotein Bolinopsin, sowie dessen Verwendung

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006108518A1 (fr) * 2005-04-13 2006-10-19 Bayer Healthcare Ag Photoproteine isolee gr-bolinopsine et utilisation de celle-ci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006497A2 (fr) * 2001-07-12 2003-01-23 Centre National De La Recherche Scientifique Photoprotéines mutees et leurs applications

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2003006497A2 (fr) * 2001-07-12 2003-01-23 Centre National De La Recherche Scientifique Photoprotéines mutees et leurs applications

Non-Patent Citations (6)

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DATABASE UNIPROT 1 October 1994 (1994-10-01), INOUYE S. ET AL.: "Clytin precursor (Phialidin)", XP002300448, Database accession no. Q08121 *
FAGAN T F ET AL: "Cloning, expression and sequence analysis of cDNA for the Ca(2+)-binding photoprotein, mitrocomin.", FEBS LETTERS. 1 NOV 1993, vol. 333, no. 3, 1 November 1993 (1993-11-01), pages 301 - 305, XP002300183, ISSN: 0014-5793 *
INOUYE S ET AL: "CLONING AND SEQUENCE ANALYSIS OF CDNA FOR THE CA2+-ACTIVATED PHOTOPROTEIN, CLYTIN", FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 315, no. 3, January 1993 (1993-01-01), pages 343 - 346, XP001180448, ISSN: 0014-5793 *
INOUYE S ET AL: "Cloning and sequence analysis of cDNA for the luminescent protein aequorin.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. MAY 1985, vol. 82, no. 10, May 1985 (1985-05-01), pages 3154 - 3158, XP002300184, ISSN: 0027-8424 *
MARKOVA SVETLANA V ET AL: "Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins.", BIOCHEMISTRY. 19 FEB 2002, vol. 41, no. 7, 19 February 2002 (2002-02-19), pages 2227 - 2236, XP002232863, ISSN: 0006-2960 *
See also references of EP1638998A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006108518A1 (fr) * 2005-04-13 2006-10-19 Bayer Healthcare Ag Photoproteine isolee gr-bolinopsine et utilisation de celle-ci

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EP1638998A1 (fr) 2006-03-29

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