WO2004022786A1 - Procede d'analyse de proteines comprenant l'utilisation d'un transfert d'energie de fluorescence par resonance - Google Patents

Procede d'analyse de proteines comprenant l'utilisation d'un transfert d'energie de fluorescence par resonance Download PDF

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WO2004022786A1
WO2004022786A1 PCT/JP2003/011302 JP0311302W WO2004022786A1 WO 2004022786 A1 WO2004022786 A1 WO 2004022786A1 JP 0311302 W JP0311302 W JP 0311302W WO 2004022786 A1 WO2004022786 A1 WO 2004022786A1
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protein
fluorescent
host
interaction
baculovirus
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PCT/JP2003/011302
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English (en)
Japanese (ja)
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Takao Hamakubo
Tatsuhiko Kodama
Kenjiro Miyano
Kazuyuki Masuda
Toshiko Sakihama
Hiroharu Tamaru
Hirotaka Ito
Atsushi Miyawaki
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Toudai Tlo, Ltd.
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Priority to AU2003261932A priority Critical patent/AU2003261932A1/en
Publication of WO2004022786A1 publication Critical patent/WO2004022786A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the present invention relates to a method for analyzing protein interaction using fluorescence resonance energy transfer. More specifically, the present invention provides for functionally expressing a protein in a baculovirus released from a host, and analyzing the structural change of the protein or its interaction with other proteins using fluorescence resonance energy transfer. About the method.
  • the baculovirus expression system utilizes a baculovirus high-expressing protein, particularly a polyhedrin gene promoter, and causes the target gene to recombine in insect cells (such as Sf9 cells). It is a system that is expressed in large quantities. A system that introduces a recombinant protein into a polyhedron gene and purifies the expressed protein.
  • the expression system of Baculo is more capable of expressing proteins even if it has a large number of hydrophobic regions such as membrane proteins, compared to expression systems using Escherichia coli or yeast.
  • Tate CG Gr is shammer R., Trends in Biotechnology 1996, 14, pp426-430, Heterologous expression of G-protein-coupled receptors; and Gris shammer R, Tate CG, Quarterly Reviews of Biophysics 1995, 28, pp315-422 , Overexpression of integral membrane proteins for structural studies).
  • Baculoviruses have another life cycle in addition to being in the nucleus, which is a polyhedral virus that has suffered from polyhedral proteins. It becomes a virus (Budded virus) and covers the cell membrane of Sf9 cells and goes out of the cells. At this time, the seven-transmembrane receptor, which had been recombined into the above-mentioned polyhedrin protein, was expressed on the cell membrane of Sf9, Recovery was reported by Loisel et al. (Loisel TP, Ansanay H, St-0nge S, Gay B, Boulanger P, Strosberg AD, Marullo S, Bouvier M., Nat Biotechnol. 1997, Nov.
  • G-protein coupled receptors also called seven-transmembrane receptors, are membranes that transmit signals into cells in response to stimuli such as hormones, light, odor or taste.
  • Tate CG Grisshammer R., Trends in Biotechnology 199b, 14, pp 426-430, Heterologous expression of G-protein— coupled receptors; and Grisshammer R, Tate CG, Quarterly Reviews of Biophysics 1995, 28, pp315 - 422, Overexpression of integral membrane proteins for structural studies
  • 7 0 0 kind about genes are present, including the [Nobi Rere receptor ( Venter JG, Adams MD, Myers EW, et al., Science 291, ppl304-1351, 2001, The sequence of the human genome) 0 Many of these are receptors for honolemon-chemokines.
  • the GPCR binds to a ligand, activates the trimeric G protein, dissociates the G a subunit of the trimeric G protein, interacts with the effector protein, and transmits a signal.
  • GPCRs maintain high affinity by coupling to G proteins, and detecting this signal is important for identifying ligands for receptors and screening for inhibitors.
  • FRET fluorescence resonance energy transfer
  • GFP green fluorescent protein
  • GFP has a relatively large molecular weight itself, the conformation of the target protein changes when incorporated, and the function may be impaired. Particularly in the case of GPCRs, complex systems such as ligand binding and interaction with G proteins are transmitted in relatively small domains, so when GFP is fused to the receptor, the function of G protein coupling is impaired. Many things to be done. In addition, since the G protein itself has various functions such as trimer formation, GDP / GTP exchange reaction, coupling with GPCR, and interaction with ⁇ effector protein, GFP is fused while maintaining the function of G protein. It was difficult.
  • An object of the present invention is to provide a method for easily detecting a protein interaction. More specifically, the present invention provides a method for easily detecting an interaction between proteins in signal transduction via a G protein-coupled receptor such as an interaction between a G protein and its effector protein. This was an issue to be solved. Another object of the present invention is to provide a method for screening a novel drug by detecting an interaction between proteins in signal transduction in the presence of the drug.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, using a technique for expressing a functional GPCR and a G protein in baculovirus, a recombinant expressing an addate cyclase with CFP introduced as a fluorescent donor.
  • a technique for expressing a functional GPCR and a G protein in baculovirus a recombinant expressing an addate cyclase with CFP introduced as a fluorescent donor.
  • a host infected with at least one type of recombinant paculovirus containing a gene encoding at least one type of protein fused with a fluorescent protein is cultured and released from the host.
  • a protein analysis method comprising expressing the protein in a baculovirus, and detecting the interaction or structural change of the protein by fluorescence resonance energy transfer.
  • At least one recombinant baculovirus containing a gene encoding the first protein fused to the first fluorescent protein and a gene encoding the second protein fused to the second fluorescent protein is used.
  • the infected host is cultured, the first protein and the second protein are co-expressed in the baculovirus released from the host, and the proteins interact with each other, and the interaction is performed by fluorescence resonance energy transfer. Detect more.
  • the first protein is a G protein
  • the second protein is a G protein eflator protein
  • the host is further infected with a recombinant baculovirus encoding a G protein-coupled receptor.
  • the host is cultured in the presence of a ligand for a G protein-coupled receptor.
  • the paculovirus released from the host is a germinated baculovirus.
  • the host is an insect cell or insect larva.
  • a method for analyzing a protein interaction in the presence of a test substance and screening for a substance that promotes or inhibits the interaction in the above-described analysis method of the present invention is provided.
  • FIG. 1 shows the results of ⁇ -Estan blot using D1-dopamine receptor expressing virus and infected Sf9 cells.
  • Lane 1 shows the wild type budding virus
  • lane 2 shows the D1-DR expressing budding virus
  • lane 3 shows the wild type Sf9 cell membrane fraction
  • lane 4 shows the Dl-DR expressing Sf9 cell membrane fraction.
  • FIG. 2 shows detection of a receptor-specific FRET phenomenon by stimulation with 100 ⁇ M dopamine.
  • D 1 / G s -Citrine / AC 6 -EC FP D 1 and G a s -Citrine, G ⁇ , G ⁇ , AC 6- ECFP coexpression baculovirus
  • the method for analyzing a protein comprises culturing a host infected with at least one type of recombinant baculovirus containing a gene encoding at least one type of protein fused with a fluorescent protein, and baculovirus released from the host.
  • the protein is expressed in a virus, and the interaction or structural change of the protein is determined by fluorescence resonance energy.
  • the analysis method of the present invention preferably comprises at least one gene containing a gene encoding the first protein fused to the first fluorescent protein and a gene encoding the second protein fused to the second fluorescent protein. Culturing a host infected with the recombinant paculovirus, co-expressing the first protein and the second protein in the baculovirus released from the host, and allowing the proteins to interact with each other; The interaction is performed by detecting the interaction by fluorescence resonance energy transfer.
  • the genes encoding the two types of proteins may be contained in the same recombinant baculovirus or may be contained in different recombinant baculoviruses. .
  • the recombinant baculovirus encoding the G protein-coupled receptor is further infected to the host, and the host is cultured in the presence of a ligand for the G protein-coupled receptor, whereby the ligand binds to the receptor. It is possible to measure signal transduction via the receptor.
  • the “G protein-coupled receptor” referred to in the present specification is also called a seven-transmembrane receptor, and transmits signals intracellularly in response to stimuli such as hormones, light, odor or taste. It is a membrane receptor that transmits to Many of the G protein-coupled receptors are receptors for the hormone ⁇ chemokines, which bind to ligands to activate the trimeric G protein, dissociate the Get subunit of the trimeric G protein, and bind to the effector protein. Propagate the signal through interaction.
  • the G protein referred to in the present invention includes a trimeric G protein.
  • the following are examples of the ⁇ subunit of the G protein.
  • Gs class Gas Gaolf 0 Gi class Gail, Gai2, Gai3, Gaol, Gao2, Gatl, Gctt2, Gagust.
  • G ctq Gall, Gal4, G ⁇ xl5, Gal6 of Gq class.
  • G12 class Gal2, Gal3 0 also form these a Sabuyunitto and trimer / 3 and gamma Sabuyunitto, respectively
  • Effector proteins of G protein include Adeele cyclase, phospholipase C] 3, and Rho GEF.
  • G as and G ai are adjuvant cyclases
  • G CK q is a phospholipase C] 3
  • G o; 12 and G ⁇ 13 are R ho GEF, respectively, effector proteins.
  • G protein-coupled receptor protein the ligand is shown in parentheses
  • specific examples of the G protein-coupled receptor protein include the following.
  • PTH receptor ratonolemon
  • MGK ⁇ mGlu 2 glutamic acid
  • GABA B gamma - Amino butyric acid
  • a first protein eg, a G protein
  • a second protein eg, a G protein effector protein
  • a first protein eg, a G protein
  • a second protein eg, an effector protein of a G protein
  • the first protein may be fused with a fluorescent donor protein and the second protein may be fused with a fluorescent receptor protein.
  • the type of the fluorescent protein used in the present invention is not particularly limited, and examples thereof include cyan fluorescent protein (CFP), yellow fluorescent protein (YEP), green protein (GFP), red fluorescent protein (REP), and blue fluorescent protein. Protein (BFP) or a mutant thereof.
  • cyan fluorescent protein, yellow fluorescent protein, green protein, red fluorescent protein, blue fluorescent protein or a mutant thereof means not only a known fluorescent protein but also a mutant thereof (for example, It is meant to include all of EFP, EYFP, EGFP, ERFP, EBFP, etc., which have enhanced protein fluorescence intensity.
  • the gene for the green fluorescent protein has been isolated and sequenced (Prasher, D. s. B. (1992), 'Primary structure of the Aequorea victoria green fluorescent protein', Gene 111: 229-233). Numerous amino acid sequences of the fluorescent protein or its variant have also been reported, for example, as described in Roger Y. Tsien, Annu. Rev. Biochem. 1998.
  • the fluorescent protein GFP
  • the yellow fluorescent protein YFP
  • a mutant thereof for example, those derived from Owang jellyfish (for example, Aequorea victoria) can be used.
  • GFP GFP
  • YFP YFP
  • mutants examples include GFP, YFP and their mutants.
  • the notation F 99 S means that the 99th amino acid residue starts with F s, and other amino acid substitutions are indicated in the same manner.
  • G F P having an amino acid mutation of F 99 S, Ml 53 ⁇ , V 163 A;
  • GFP having an amino acid mutation of S 65 T;
  • GFP having an amino acid mutation of F 64 L, S 65 T;
  • GFP having an amino acid mutation of S65T, S72A, N149K, Ml53 ⁇ , I167T;
  • GFP having an amino acid mutation of S 202 F, T 203 I;
  • G F P having an amino acid mutation of T 203 I, S 72 A, Y 145 F;
  • GFP having an amino acid mutation of S65G, S72A, T203F (YFP); GFP having an amino acid mutation of S65G, S72A, T203H (YFP); S65G, V68L, Q69K , GFP having an amino acid mutation of S 72 A, T 203 Y (E YF PV 68 L, Q 69 K);
  • GFP having an amino acid mutation of S65G, S72A, T203Y
  • GFP having an amino acid mutation of S65G, S72A, K79R, T203Y
  • the nucleotide sequence of the gene encoding the fluorescent protein used in the present invention is known.
  • the gene encoding the fluorescent protein may be a commercially available gene.
  • EGFP vector, EYFP vector, ECFP vector, EBFP vector, and the like which are commercially available from Clontech, can be used.
  • Combinations of the fluorescent donor Z fluorescent receptor that can be used in the present invention include, but are not limited to, CFPZYFP or BFP / GFP.
  • CFPZYFP CFPZYFP
  • BFP / GFP BFP / GFP.
  • ECFP can be used as a fluorescent donor
  • citrine a modified YFP Q69M
  • Construction of a gene in which a fluorescent protein encodes a fusion protein can be performed by a conventional method known to those skilled in the art. It can be performed using a genetic recombination technique.
  • At least one type of recombinant Pacuviruses containing a gene encoding the above-mentioned fluorescent fusion protein is used.
  • Baculovirus a virus that causes disease by infecting insects, is an enveloped virus that has a circular double-stranded DNA as a gene and is susceptible to insects such as Lepidoptera, Hymenoptera, and Diptera.
  • the nuclear polyhedrosis virus is a group of viruses that make large amounts of inclusion bodies called polyhydra (polyhydra) in the nucleus of infected cells.
  • Polyhedra are composed of a polyhedrin protein with a molecular weight of 31 kDa and are produced in large quantities at the late stage of infection, in which many virus particles are embedded. Polyhedra are essential for the virus to survive in nature, but are not necessary for virus growth itself, so even if a foreign gene that is to be expressed in place of the polyhedron gene is inserted, the virus can be transmitted without any problem. And proliferate.
  • viruses used in the present invention include viruses such as Autographs calif ornica NPV (Ac NPV) of NPV subfamily and Bombyx mori NPV (BmNPV) of silkworm. Can be used as a vector.
  • viruses such as Autographs calif ornica NPV (Ac NPV) of NPV subfamily and Bombyx mori NPV (BmNPV) of silkworm.
  • Ac NPV Autographs calif ornica NPV
  • BmNPV Bombyx mori NPV
  • Ac NPV hosts include Spodoptera frugiperda cells (Sf cells), and BmNPV hosts (infected, passaged cells) include BmN4 cells. . Since Sf cells have a higher proliferation rate than BmN4 cells, etc., and AcNPV has the ability to infect human hepatocytes and human fetal kidney cells, etc., AcNPV-based vectors are preferable.
  • Spodoptera Frugiperda cell lines Sf9 and Sf21 have been established from the ovarian tissue of S. frugiperda larvae, and are available from Invitrogen, Pharmingen (San Diego, CA), ATCC, or the like. In addition, live insect larvae can be used as host cell systems.
  • the method of constructing the recombinant virus used in the present invention may be performed according to a conventional method. For example, it can be performed in the following procedure.
  • a gene of a protein to be expressed is inserted into a transfer vector to construct a transfer vector.
  • the overall size of the transfer vector is generally about several kb to 10 kb, of which about 3 kb is a plasmid-derived backbone, which is used for the resistance of an antibiotic resistance gene such as ampicillin to the initiation of bacterial DNA replication. Contains the signal.
  • the region 5 and region 3 of the polyhedron gene are each several kb each, and when the transfection described below is performed, the target gene is inserted between these sequences. And homologous recombination between the polyhedron gene.
  • the transfer vector preferably contains a promoter for expressing the protein gene. Examples of the promoter include a polyhedron gene promoter, a 10 gene promoter, and a capsid gene promoter.
  • the type of transfer vector is not particularly limited.
  • Specific examples of the transfer vector include Ac NPV transfer vectors, such as pEVmXIV2, pAcSGI, pVL1392 / 1393, pAcMP2 / 3, pAcJP1, AcUW21, AcDZ 1, Blue B ac II pAc UW51, AcAB3, pAc360, pBlueeBacHis, pVT-Bac33, pAcUWl, pAcUW42Z43, etc., and BmNPV transfer As vectors, ⁇ 283, ⁇ 5, ⁇ 30, ⁇ 1, ⁇ > ⁇ 2, pBK3, pBK52, pBKb lue, pBKb1ue2, pBF series (Funakoshi, Fujisawa Pharmaceutical Co., Ltd. Etc.).
  • the above-mentioned recombinant transfer vector is mixed with the virus and then transferred to a cultured cell used as a host. Transfer vector from the recombinant transfer vector to the viral genomic DNA. causess homologous recombination to construct a recombinant virus.
  • the cultured cells used as hosts include the above-mentioned hosts, and are usually insect cultured cells (Sf9 cells, BmN cells, etc.). Culture conditions are appropriately determined by those skilled in the art.Specifically, when Sf9 cells are used, culture can be performed at around 28 ° C in a medium containing 10% fetal serum. preferable.
  • the recombinant virus thus constructed can be purified by a conventional method, for example, plaque assay.
  • the recombinant virus produced in this manner cannot replace the inserted or inserted foreign DNA into the gene region of the polyhedron protein of the nuclear polyhedrosis virus, and cannot form a polyhedron. It can be easily distinguished from a replacement virus.
  • the above-described recombinant virus is infected to an appropriate host (cultured cells such as Spodoptera Frugiperda cell lines Sf9 and Sf21, or insect larvae), and after a certain period of time (for example, after 72 hours).
  • Extracellular budding virus (BV) can be recovered from the culture supernatant by a separation operation such as centrifugation.
  • the recombinant baculovirus may be infected with only one kind, or may be co-infected with a combination of two or more kinds of recombinant baculoviruses.
  • the extracellular budding baculovirus can be collected, for example, as follows. First, the culture of the infected cells is centrifuged at 500 to 1,000 g, and the supernatant containing the extracellularly germinated paculovirus is recovered. The supernatant can be centrifuged at about 30,000 to 500,000 g to obtain a precipitate containing extracellular budding baculovirus.
  • the budding baculovirus recovered as described above contains a fusion protein fused with a fluorescent protein.
  • the FRET phenomenon of the budding baculovirus prepared as described above can be explained by, for example, using a 410 nm purple laser diode (Nichia Corporation ) Can be analyzed by exciting the light to a light source and measuring the wavelength longer than 435 nm using a GG435 long-pass filter (Control Optics). Fluorescence can be measured, for example, using a 30 cm spectroscopy system (3001ines; It can be analyzed by Blaze 500nm) (Acton research) and analyzed by liquid nitrogen cooled CCD camera (512x512pixel; 16bit depth) (Princeton Instrument). When CFP ZYFP is used as the combination of the fluorescent donor / fluorescent acceptor, the ratio of the fluorescence intensity can be calculated by setting the wavelength integration ranges of CFP and YFP to 465-495 nm and 510-540 nm.
  • the present invention further provides the above-described protein analysis method, wherein the interaction of the protein in the presence of a test substance is analyzed using fluorescence resonance energy transfer, and a substance that promotes or inhibits the interaction is screened. I will provide a.
  • Test substances to be screened include, for example, peptides, polypeptides, synthetic compounds, fermented microorganisms, extracts from living organisms (including plant or animal tissues, microorganisms, or cells), or extracts thereof.
  • Libraries. Libraries include synthetic compound libraries (such as combinatorial libraries) and peptide libraries (such as combinatorial libraries).
  • the chemicals to be screened may be natural or synthetic, and even if a single candidate chemical is tested independently, a mixture of several candidate chemicals (such as a library) May be tested. It is also possible to screen a fractionated mixture, such as a cell extract, to isolate a substance having the desired activity by repeating the fractionation.
  • test substances can promote or inhibit the interaction between the membrane receptor protein and the ligand, the interaction between the membrane receptor protein and the G protein, or the interaction between the G protein and its effector protein.
  • it is an expected substance.
  • the screening method of the present invention it is possible to screen an inhibitor or an activating drug for a membrane-type receptor protein, an inhibitor or an activating drug for a G protein, or an inhibitor or an activating drug for a G protein effector. It is possible. Substances that promote or inhibit such protein interaction, which are obtained by the screening method of the present invention, are also within the scope of the present invention.
  • Example 1 Preparation of recombinant baculovirus expressing dopamine receptor D1
  • the full-length human dopamine receptor D1 (DR-D1) cDNA obtained by amplifying the human fetal brain cDNA by PCR was inserted into pBlueBacHis2C (Invitrogen) to prepare pBluBac-His-DR-Dl.
  • Sf9 cells (Invitrogen) are grown in complete medium (Grace's supplemented media (GIBCO BRL) containing 10% fetal serum (Sigma), 100 units / ml penicillin and 100 ⁇ g / ml streptomycin) in 27 ° C. The cells were subcultured on C to a 10 cm diameter tissue.
  • the precipitate was suspended in PBS and centrifuged at 800 xg for 10 minutes to remove aggregates.
  • the precipitate obtained was centrifuged again at 40,000 xg for 25 minutes, and the precipitate obtained was suspended in PBS and the budding virus fraction (BV fraction) was removed. Minute).
  • reaction volume was adjusted to 500 ⁇ l by adding the DR-DlBV fraction, and the reaction was carried out at room temperature for 90 minutes.
  • the reaction phthalate - ⁇ - heptyl / phthalate Jiotachiru 1 layered over 1 mixture 500 mu 1, 15, 000 x g, 10 minutes, and centrifuged at room temperature, was recovered as a precipitate virus. The precipitate was washed three times with a binding buffer, air-dried, and the amount of 3 H-dopamine bound to the receptor was calculated by measuring the amount of tritium contained in the precipitate with a liquid scintillation counter.
  • G proteins are activated by specific ligand binding to coupled receptors, interact with various enzymes collectively called effector proteins after activation, and regulate the activity of these enzyme molecules.
  • Adenylate cyclase is an effector protein that increases cyclic AMP (cAMP) production by interacting with activated Gs-like G proteins. It has been reported that the dopamine receptor couples with the Gs-like G protein, and the Gs-like G protein activated by specific binding of dopamine interacts with adenylate cyclase to increase its activity. . Also, dopamine does not show specific binding to platelet activator receptor (PAFR).
  • PAFR platelet activator receptor
  • the Overlap extension PCR was the human Ga s cDNA and Citrine cDNA as a template DNA, a cDNA sequence of Citrine between cDNA of 127D and 128F of Ga s Purchased.
  • SGGGGS peptide chain cDNA was added as a linker to the N- and C-terminals of Citrine, respectively.
  • Citrine inserted Ga s cDNA that was created is incorporated to pBlueBac4.5 vector (Invitrogen).
  • the cloned AC VI cDNA was incorporated into pECFP-Nl plasmid (Clontech), and the ECFP gene was fused to the 3 ′ end of the AC VI gene. Furthermore, this ECFP-fused ACVI cDNA was inserted into pBlueBac4.5 vector (Invitrogen).
  • the preparation method of the recombinant baculovirus described in Example 1 (1) Citrine inserted a s - a (a s Citrine) recombinant baculovirus Contact Yopi ECFP fused AC VI (AC VI- ECFP) recombinant baculovirus Prepared.
  • a PAF receptor (PAFR) recombinant baculovirus was prepared by incorporating the PAFR cDNA into pBlueBacHis2A and producing the recombinant baculovirus described in Example 1 (1).
  • PAFR PAF receptor
  • the fluorescence of the germinated paculovirus prepared in each combination was measured using a 410-nm purple laser diode (Nichia Corporation ) Was excited by a light source, and the measurement was performed on the longer wavelength side than 435 nm using a GG435 long-pass filter (Control Optics).
  • the respective wavelength integration ranges of CFP and YFP were set to 465-495 nm and 510-540 nm, and the ratio of the fluorescence intensity was calculated.
  • a novel drug can be screened by detecting an interaction between proteins in signal transmission in the presence of the drug.

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Abstract

L'invention concerne un procédé permettant une détection aisée de l'interaction d'une protéine, et plus particulièrement un procédé d'analyse de protéines qui consiste à mettre en culture un hôte infecté avec au moins un baculovirus recombiné contenant un gène codant pour au moins une protéine hybridée avec une protéine fluorescente, à exprimer la protéine libérée par l'hôte dans le baculovirus, et à détecter l'interaction de la protéine ou sa modification de structure au moyen du transfert d'énergie de fluorescence par résonance.
PCT/JP2003/011302 2002-09-04 2003-09-04 Procede d'analyse de proteines comprenant l'utilisation d'un transfert d'energie de fluorescence par resonance WO2004022786A1 (fr)

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US9314450B2 (en) 2011-01-11 2016-04-19 Dimerix Bioscience Pty Ltd. Combination therapy

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EP2853267B1 (fr) 2007-09-21 2016-12-07 The Regents of the University of California Interféron ciblé manifestant des activités apoptotiques et anti-tumorales puissantes
JP5330059B2 (ja) * 2009-03-31 2013-10-30 三井造船株式会社 Ｆｒｅｔプローブ、発現ベクター、細胞、およびｆｒｅｔ測定方法
WO2014089354A1 (fr) 2012-12-07 2014-06-12 The Regents Of The University Of California Interféron ciblant l'antigène cd138 présentant de puissantes activités apoptotiques et anti-tumorales
WO2014194100A1 (fr) 2013-05-29 2014-12-04 The Regents Of The University Of California Fusions anti-cspg4 et interféron utilisables en vue du traitement de tumeurs malignes

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US9314450B2 (en) 2011-01-11 2016-04-19 Dimerix Bioscience Pty Ltd. Combination therapy
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