WO2007058454A1 - Systeme permettant de detecter les interactions moleculaires - Google Patents

Systeme permettant de detecter les interactions moleculaires Download PDF

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Publication number
WO2007058454A1
WO2007058454A1 PCT/KR2006/004772 KR2006004772W WO2007058454A1 WO 2007058454 A1 WO2007058454 A1 WO 2007058454A1 KR 2006004772 W KR2006004772 W KR 2006004772W WO 2007058454 A1 WO2007058454 A1 WO 2007058454A1
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construct
detecting
protein
label
magnetic
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PCT/KR2006/004772
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English (en)
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Tae Kook Kim
Yong-Weon Yi
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Cgk Co., Ltd.
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Publication of WO2007058454A1 publication Critical patent/WO2007058454A1/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
    • G01N33/53—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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • the present invention relates to a method for detecting interactions, in vitro and/or in vivo, between interest molecules, such as bioactive molecules. Further, the present invention relates to a method for detecting interactions, in vitro and/or in vivo, between interest molecules, such as bioactive molecules, and for screening target molecules.
  • a bioactive chemical compound refers to a chemical compound, which binds to biomolecules such as proteins, nucleic acids, mono-/oligo-/poly- saccharides or lipids and it is configured to control the function or biological activity thereof, in vivo.
  • a bioactive chemical compound is extracted from an organism or prepared by chemical synthesis.
  • antibiotics for example, "CYCLOSPORINE A” (Novartis AG) and "FK506" (Fujisawa), which are used for reducing immune rejection following an organ transplantation, were isolated from a microorganism, a plant or a marine organism.
  • Such a natural or synthetic bioactive chemical compound is developed as a new drug through pharmacological activity tests and clinical tests thereof, while employing an animal model and a human model.
  • a protein performs its biological functions in vivo by binding to other proteins.
  • the complement proteins interact with each other, and a bioactive chemical compound binds specifically to a specific portion of the 3 -dimensional protein structure.
  • the interaction between two proteins strongly implies that they are functionally related.
  • the bioactive chemical compound binding to a specific portion of a protein especially relevant to diseases has potential as a therapeutics which diagnoses, prevents, treats or alleviates a disease by controlling the activity of the protein. Accordingly, various methods for screening bioactive molecules as drug candidates and detecting novel target proteins as therapeutic targets have been studied, wherein the methods detecting the interaction between a "bait" whose function and feature are known and a "prey" which is an interaction partner of the bait.
  • target protein helps to understand the biological pathway and signal transduction system, and provides information on fundamental cellular regulation, physiology and/or mechanism of disease. Accordingly, such information is a powerful tool for, e.g., new drug developments, modification of existing drugs and discovery of novel pharmaceutical usage via detecting and screening of bioactive molecules binding to the target protein(s).
  • the methods for detecting interactions between molecules in vitro are as follows: conventional biochemical method of, for example, cross-linking, radiolabeled ligand binding, X-ray crystallography and affinity chromatography, affinity blotting, immunoprecipitation and mass spectrometry (E. M. Phizicky and S. Fields, Microbiol. Rev., 59: 94-123, 1995; A. R. Mendelsohn and R. Brent, Science, 284: 1948- 1950, 1999).
  • the above-mentioned conventional methods require protein production and isolation processes therefor, which in turn demand much effort.
  • these methods are to be carried out in vitro, it cannot provide the exact information on in vivo interactions.
  • phage display cloning (Sche, P.P., McKenzie, K.M., White, J.D., and Austin, DJ. 1999, Display cloning: functional identification of natural product receptors using cDNA-phage display. Chem. Biol. 6, 707-716), fluorescence resonance energy transfer (FRET; Moshinsky DJ, Ruslim L., Blake RA, Tang F., A widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype. J. Biomol. Screen.
  • the yeast two-hybrid system is characterized in that a gene expression is performed through a reconstruction of a transcriptional factor obtained by interactions of two expressed proteins.
  • the expressed proteins expected to interact with each other have been cloned into a DNA binding domain (DBD) and a transcriptional activation domain (TAD), respectively, through recombinant DNA technology, and then the proteins are allowed to be expressed within a yeast cell (Fields and Song, Nature 340: 245-246. 1989).
  • DBD DNA binding domain
  • TAD transcriptional activation domain
  • This system can be conducted in vivo and offers a relatively easy test for conducting. Further, since interactions between two recombinant proteins can be monitored through activity of a reporter gene, sensitivity control is possible depending on the selections of kind and nature of the reporter gene.
  • the system can be used to detect domains or amino acids that are critical to interactions and a measurement of binding affinity is possible as well.
  • this system is unreliable for genomes that are big and complex and its interpretation may become difficult, implementing the same to humans may also be difficult.
  • the system should be selected based on the original location of the protein in a cell, and a transcription activity can be affected depending on the cell metabolism and growth thereof. Further, it is difficult to obtain a high quality library, and the hybrid proteins can be toxic.
  • the Y3H system is characterized in that detections on interactions between bioactive chemical compounds and proteins are conducted in living cells.
  • the Y3H system includes three hybrid molecules, i.e. a DNA-binding protein (DBD) fused with a ligand binding domain (LBD) of a bioactive chemical compound, a hybrid protein containing a transcriptional activation domain (TAD) fused with a second LBD, and a bivalent hybrid molecule.
  • DBD DNA-binding protein
  • LBD ligand binding domain
  • TAD transcriptional activation domain
  • the Y3H like the Y2H, is limited in that it should be conducted in a simple cell such as a yeast cell.
  • yeast cell has a drawback in that it has genetically a lower permeability for bioactive chemical compound when compared to mammalian cells.
  • the phage display libraries have been developed as a method for screening an antibody binding specifically to an antigen by using antibody libraries. Recently, a method which identifies an interaction map via the two-hybrid system has been published. Here, the map is created by screening candidate proteins with bioinformatics technology after finding via peptide libraries a consensus sequence that binds to a specific protein (Allen JB, Walberg MW 9 Edwards MC, Elledge SJ., Finding prospective partners in the library: the two-hybrid system and phage display to find a match, Trends Biochem Sci. 1995 Dec; 20(12): 511-6). However, since its fundamental process is performed in vitro, it has a drawback of having a high false positive rate.
  • bioinformatics method which predicts interactions between proteins. This method predicts interactions between proteins through molecular phylogenies or multiple sequence alignment, based on the database of genome sequence information. Also, there are relatively newly developed virtual screening methods such as structural genomics, molecular fingerprint, and various cluster analyzing methods.
  • Ding S. et. al. identified a chemical compound, which induces a P19 stem cell to differentiate to a nerve cell, by screening purine molecule library using affinity chromatography (Ding, S., T.Y.H., Brinker, A., Peters, E.C., Hur, W., Gray, N.S., and Schultz, P. G. 2003, Synthetic small molecules that control stem cell fate. Proc. Natl. Acad. Sci. USA 100, 7632-7637), as a biochemical method.
  • affinity chromatography Ding, S., T.Y.H., Brinker, A., Peters, E.C., Hur, W., Gray, N.S., and Schultz, P. G. 2003, Synthetic small molecules that control stem cell fate. Proc. Natl. Acad. Sci. USA 100, 7632-7637
  • the potent relationship between phenotype and genotype is the biggest advantage of this method. That is, one hit identified using this method provides directly a gene clone to a target protein (Jaeger, S., Eriani, G, and Martin, F. 2004, Results and prospects of the yeast three-hybrid system. FEBS Lett. 566, 7-12).
  • a novel target protein candidate can be used in the fused form as a part of a multivalent protein complex. This system, however, is allowed to be used only in yeast.
  • the chemical genomics which uses a bioactive chemical compound in order to determine the effect of gene mutation on a cell, identifies an active chemical compound from a group of chemical compounds or from a combinatorial library (Schreiber, S. L. 2003, The small molecule approach to biology. Chem. & Eng. 1199 News 81, 51-61; Crews, CM., and Splittgerber, U. 1999, chemical genetics: exploring and controlling cellular processes with chemical probes. Trends Biochem. Sci. 24, 317-320).
  • a report construct having a promoter inducing the expression of a GFP or a commercial marker will be included in a cell, in order to confirm the expression of a certain gene or a group of genes.
  • this method has also two significant problems: first, the chemical genomics is not suitable to detect a bioactive chemical compound which functions at nanomolar concentration; secondly, only a couple of active molecules can be detected from hundreds of thousands of materials, using this method.
  • most of the conventional technologies for detecting the interaction between proteins or for detecting the interaction between a protein and a bioactive chemical compound can be carried out only in limited kinds of cells, such as, yeast or bacteria cell. And most of the conventional technologies should be carried out in vitro. Since the most of the conventional technologies require gene manipulation and expression process, it is difficult to implement the above technologies as they are limited by the size of DNA. Further, the higher false positive rate due to other parameters lowers reliability of the result.
  • the inventors of the present invention have developed a new method for detecting molecular interactions of bioactive molecules. Such method is completed by the following order: after introducing into a cell a bioactive chemical compound attached to a localizer showing magnetism to be shifted under magnetic field and other bioactive chemical compounds attached to a label material, a magnetic field is applied to the above cell and the location of the label shifted under magnetic field is detected, followed by detection and verification of the molecular interaction of bioactive molecules.
  • An example of the methods has been published as International Patent Application No. PCT/KR2005/002352. Despite this, a constant improvement is still possible like any other outstanding technologies.
  • a bioactive chemical compound is required to be bonded to a localizer such as a magnetic particle.
  • the bioactive chemical compound is a protein or a molecule that binds to proteins
  • the bioactive chemical compound being a protein or a molecule that binds to proteins
  • a complex of the localizer and the bioactive chemical compound is also necessary.
  • the object of the present invention in general, is to provide a real-time detection method for detecting interactions between interest molecules, such as biomolecules, in vivo (i.e., in cell, in tissue or in body) and in vitro, and for screening target molecules.
  • this invention is for providing a method for directly detecting interactions, in vivo and in vitro, between "the first detecting material" of a certain biomolecule and "the second detecting material” of a biomolecule to be detected, valuated and/or screened.
  • Another object of the present invention is to provide a method for directly detecting interactions, in vivo and in vitro, between "the first detecting material” and “the second detecting material", and for screening a target molecule.
  • Still another object of the present invention is to provide a method for indirectly detecting interactions, in vivo and in vitro, between "the first detecting material” and “the second detecting material” by monitoring the change in cellular morphology or function.
  • the inventors of the present invention designed two constructs. More specifically, the first construct comprises a magnetic protein, which is translocated by a magnetic force, and the first detecting material, such as a biomolecule to be analyzed, wherein the first detecting material is attached to the magnetic protein directly or indirectly by the use of, for example, a linker.
  • the magnetic protein and the first detecting material of biomolecule can be provided as a fused form.
  • the first construct can be provided as a structure, which are formed by an indirect binding of at least one of the first detecting materials of biomolecules and magnetic proteins to which probes are fused to help to recognize the biomolecules.
  • the second construct includes one or more second detecting materials, such as biomolecules, to which at least one label is attached for detection.
  • the label and the biomolecule to be analyzed can be provided in a directly fused form.
  • the second construct can be provided as a structure, which are formed by an indirect binding of at least one of the second detecting materials, e.g. biomolecules, and labels to which the probes are fused to help recognize the biomolecules.
  • a detection of the complex formed by the interaction of the first detecting material and the second detecting material is performed by approaching them near enough to interact with each other in the same field or system, followed by applying externally the magnetic force and detecting the label which has changed in location or motion by employing a suitable apparatus.
  • the change of the location or motion can be monitored by the use of, for example, an optical method employing, such as microscope, and can be monitored by a scanner, radioactive label detecting device, fluorescence polarization reader (FP reader), spectrophotometer, MRI (magnetic resonance imaging), SQUID, fluorescence detector and luminescence detector, etc.
  • an optical method employing, such as microscope, and can be monitored by a scanner, radioactive label detecting device, fluorescence polarization reader (FP reader), spectrophotometer, MRI (magnetic resonance imaging), SQUID, fluorescence detector and luminescence detector, etc.
  • the method for detecting molecular interactions of the present invention comprises the steps of: i) providing a first construct of a binding complex of a first detecting material and a magnetic protein which is translocated by an externally applied magnetic force; ii) providing a second construct comprising a second detecting material to which a label is attached for detection; iii) approaching the first construct and the second construct, in the same field or system, near enough to interact with each other; and iv) applying an external driving force followed by detecting the label which has changed in location or motion.
  • the method for screening a target molecule of the present invention comprises the steps of: i) providing a first construct of a binding complex of a first detecting material and a magnetic protein which is translocated by externally applied magnetic force; ii) providing a second construct comprising a second detecting material to which a label is attached for detection; iii) approaching the first construct and the second construct, in the same field or system, near enough to interact with each other; iv) applying external magnetic force followed by detecting the label changed in location or motion; and v) isolating and identifying the molecule from the second construct.
  • the screening of the target molecule can be further carried out using a conventional method, for example, RT-PCR, genomic DNA PCR or using Mass Spectroscopy.
  • the method of the present invention comprises the steps of: i) providing a first construct of a binding complex of a first detecting material and a magnetic protein which is translocated by an externally applied magnetic force; ii) providing a second construct comprising a second detecting material to which a label is attached for detection; iii) providing a third construct, which mediates interactions between the first and the second constructs and includes a target molecule; iv) approaching the three constructs, in the same field or system, near enough to interact with each other; v) applying an external magnetic force followed by detecting the label having changed in location or motion; and vi) isolating and identifying the molecule from the third construct.
  • the screening of the target molecules can be further performed by a conventional method, for example, RT-PCR, genomic DNA PCR or a Mass Spectroscopy.
  • the molecule to be detected in the first, the second or the third construct can be provided as a library.
  • the first construct of the present invention is prepared by binding a specific molecule such as a biomolecule to a metal storage protein (referred to as 'magnetic protein') having magnetism influenced by the externally applied magnetic force.
  • the above magnetic protein can be provided by being bonded to two or more specific molecules.
  • the second construct is prepared by attaching a label configured to detect partner molecules, i.e. other biomolecules, which interact with biomolecules among the first construct.
  • the interest target molecule can be detected in real-time after the first construct and the second construct are introduced into the same cell followed by applying a magnetic field as a driving force and by determining the translocated label. Further, in the present invention, the detection can be performed by changing the strength of the magnetic field. And the detection by comparison is performed by a negative control that does not show any change in location or motion, or using a positive control that shows change in location or motion, while varying the strength of the external magnetic field.
  • the present invention comprises the steps of: i) providing a specific probe by binding it to a magnetic protein; ii) introducing the magnetic protein having the probe bonded thereto into a cell containing a bioactive molecule having a label attached thereto; iii) introducing other bioactive molecule, which is to be bonded to the probe, into the cell; iv) moving the magnetic protein by applying a magnetic field to the cell; v) monitoring location of the label; and vi) isolating and identifying the bioactive molecule from the cell in which the label has changed in location.
  • a system for detecting interactions of molecules in accordance with the present invention includes: a reactor; a first construct comprising a magnetic protein, which is translocated by an externally applied magnetic force, and a first detecting material to which the magnetic protein is bonded, or comprising a base sequence for encoding the magnetic protein and the first detecting material; and a second construct comprising a label for detection and a second detecting material to which the label is bonded, or comprising a base sequence for encoding the label and the second detecting material.
  • the above system is characterized in that, when introducing the first and the second constructs into the reactor for an interaction therebetween and applying an externally applied magnetic force thereto, the label is varied in location or motion according to an interaction between the first and the second detecting materials.
  • the method of this invention can be carried out in an organism, a tissue or a cell of zebra fish, C-elegans, yeast, fly or frog.
  • the first construct, the second construct and the third construct are introduced into a cell by the use of, e.g., lipid (or liposome) or the binding complex thereof; or by electroporation or magnetofection.
  • this invention can be carried out in a culture plate/dish.
  • microarrayed cells can be employed for this invention.
  • the externally applied driving force in the present invention depends on features, such as the physical, chemical, biological and electrostatic feature, of the magnetic protein of the first construct.
  • bioactive molecule is understood as including all the materials, which represent biological activities in vivo, such as nucleic acid, mono- /oligo-/poly-nucleotide, protein, mono-/oligo-/poly-peptide, amino acid, mono-/oligo-
  • the "bait” refers to a bioactive molecule used for detecting interaction with other bioactive molecule.
  • the "prey” refers to a bioactive molecule to be detected or screened, which is an interaction partner of the "bait”.
  • a target molecule refers to the bait or the prey, which interacts with the prey or the bait, respectively. Also, the target molecule means all the materials to be identified, which activate or induce the interactions between the bait and the prey or which block or inhibit the interactions between the bait and the prey.
  • the "magnetic protein” refers to a metal storage protein having magnetism influenced by a magnetic force externally applied thereto.
  • a metal storage protein includes ferritin, bacterioferritin, ferritin-like protein, magnetosome, metal binding protein, and so on.
  • the "detecting material" refers to a material to be assayed and the first detecting material refers to one or more detecting materials that are directly or indirectly bonded to the first construct. Further, the second detecting material refers to one or more detecting materials that are directly or indirectly bonded to the second construct. The third detecting material is to indicate one or more detecting materials that are directly or indirectly bonded to the third construct.
  • the externally applied driving force means all types of forces which result in a translocation or motion of the magnetic protein defined above.
  • the external driving force includes, for example, electro-magnetic force, magnetic force and so on.
  • the label of the second construct which is used for detection comprises a fluorescent material, which emits fluorescence by itself or develops fluorescence by the interaction with the first construct or other materials, for example, fluorescent dye, such as, FITC and rhodamine, etc; fluorescent protein, such as, GFP, YFP, CFP and RFP, etc.; tetracystein motif; and fluorescent nanoparticle.
  • the label of the second construct comprises a luminescent material, which emits luminescence by itself or develops luminescence by the interaction with the first construct or other materials, for example, luciferase.
  • the label of the second construct comprises radio-active label, such as 3 P, S, H and C, etc.
  • a binding between a bioactive molecule and a magnetic protein, or a binding between a bioactive molecule and a label includes, for example, a physical, chemical, electrostatic or biological direct or indirect binding.
  • the bioactive molecule can be attached to the magnetic protein or the label by the above binding.
  • the probe of the first construct or the second construct, which is used for detection comprises, for example, an antibody, protein, protein domain, protein motif and peptide, etc.
  • the probe which binds to the bioactive molecule by chemical, physical, biological or electrostatic binding, can combine the bioactive molecule and the magnetic protein or combine the bioactive molecule and the label, directly or indirectly.
  • the detection of a complex formed by a molecular interaction can be performed by reacting, in the same field or system, a first construct comprising a bioactive molecule and a magnetic protein of which location is changed according to magnetism externally applied thereto; and a second construct comprising another bioactive molecule to which a label is attached for detection, and detecting the change of location or motion of the label under magnetic field.
  • translocation of the complex can be detected by changing the strength of the magnetic field.
  • the present invention can be used for discovering a novel pharmaceutical use from existing drugs or improving the pharmaceutical activities of the existing drugs as well as in detecting and screening a target protein or a new drug candidate, without damaging the interest cell and without any limitation in size of the target molecule for detection, in vitro and in vivo.
  • Fig. 1 is a schematic drawing of the present invention.
  • Fig. 2 is a schematic drawing showing a construction of the present invention, which is for detecting an interaction between a first and a second construct.
  • Fig. 3 is a schematic drawing showing a method for detecting the interaction between the first and the second construct, which is mediated by a third construct.
  • Fig. 4 is a schematic drawing showing a method for detecting an interaction between a first and a second detecting material by using the third construct, the third construct being a hybridized ligand having a probe-binding factor and a detecting material bonded therewith.
  • Fig. 5 is a confocal microscope image showing a magnetic protein translocated by an externally applied magnetic force.
  • Fig. 6 is a confocal microscope image showing a magnetic protein translocated by an externally applied magnetic force, where a non-FAC-treated cell and a cell where only a green fluorescent protein is expressed are used for a negative control.
  • Fig. 7 is a confocal microscope image confirming a translocation of an EYFP-
  • Fig. 7 is a confocal microscope image confirming a translocation of an mRFP- ⁇ TrCP protein caused by the binding of an I ⁇ B ⁇ protein and a ⁇ TrCP protein, the binding being resulted from a TNF ⁇ treatment.
  • Fig. 8 is a confocal microscope image confirming a translocation of an EGFP-
  • FRB protein caused by the binding of a FKBP 12 protein and a FRB protein, the binding being induced by the rapamycin treatment.
  • Fig. 9 is a confocal microscope image showing the result of an mRFP-I ⁇ B ⁇ protein translocated after an IKK ⁇ protein activated by a TNF ⁇ treatment is combined with an I ⁇ B ⁇ protein which is a substrate of the IKK ⁇ protein Mode for the Invention
  • Ferritin originated from human or bacterioferritin originated from bacteria has been conventionally used as a magnetic protein.
  • the human ferritin is a spherical- shaped protein complex composed of two kinds of twenty-four subunits. It has an outside diameter of about 12 run and an inner diameter of about 9 nm, and includes more than 2500 iron atoms (Chasteen, N.D. and Harrison, P.M. 1999, Mineralization in ferritin: an efficient means of iron storage. J. Struc. Biol. 126, 182-194).
  • Human ferritin genes, FTHl (GenBank Ace. No. BCOl 3724) and FTL (GenBank Ace. No. BCO 16346) are purchased from Open BioSystems Inc. of U.S.
  • pBluescript II SK(+) (purchased from Stratagene) was used as a cloning vector.
  • the IRES sequence used for producing a double-expression vector employs pIRES2-EGFP (purchased at Clontech).
  • pcDNA3.1/Zeo(+) (purchased from Invitrogen) was used as an expression vector for a mammalian cell.
  • EGFP pEGFP-Nl; purchased from Clontech
  • mRFP Cambell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A. and Tsien, R. Y. 2002, Amonomeric red fluorescent protein.
  • a primer having an Xho I linker 5'-ATA TAC TCG AGC CAC CAT GAG CTC CCA GAT TCG TCA GA-3' (SEQ ID No. 3) and another primer having an Xba I/Apa I linker, 5'-GAT CCT CTA GAG GGC CCT TAG TCG TGC TTG AGA GTG AGC-3' (SEQ ID No. 4) are used to amplify by PCR the CDS of a FTL gene.
  • a pcDNA-FTHl vector and a pcDNA-FTL vector are produced.
  • a primer having an EcoR I linker 5'-AAC CGG AAT TCC CAC CAT GGT GAG CAA GGG CGA GGA G-3' (SEQ ID No.
  • a pB SII-FTHl vector and a pBSII-FTL vector are produced.
  • 5'-CCT AGT CTA GAC CAC CAT GGC CTC CTC CGA GGA CGT C-3' (SEQ ID No.
  • a GFP-FTHl gene and a GFP-FTL gene by processing a pBioM AGIC-GFP- FTHl vector and a pBioMAGIC-GFP-FTL vector with EcoR I and Xba I restriction enzymes, followed by cloning the genes into the pBioMAGIC-mRFP-FTL vector and the pBioMAGIC-mRFP-FTHl vector, respectively, a pBioMAGIC-GFP-FTHl -mRFP- FTL vector and a pBioMAGIC-GFP-FTL-mRFP-FTHl vector are produced.
  • a cell line of human cervical cancer a HeLa cell (purchased from ATCC) is incubated with DMEM (purchased from Invitrogen) containing 10% Fetal Bovine Serum (FBS, purchased from Invitrogen). After subculturing a HeLa cell at a rate of 4000 cells/well in a 16-well chambered slideglass (purchased from Nunc), the cell is incubated at 37 ° C for 24 hrs in a CO 2 incubator. After mixing the pBioMAGIC-GFP- FTHl vector and the pBioMAGIC-mRFP-FTHl vector, the resulting mixture is introduced into the HeLa cell by using Lipofectamine 2000 (a product name: purchased from Invitrogen).
  • each of the pBioMAGIC-GFP-FTHl vector and the pBioMAGIC-mRFP-FTL vector is introduced into the HeLa cell by using the Lipofectamine 2000. Further, a pEGFP-Nl vector is introduced into the HeLa cell as a negative control. After incubating the DNA-introduced HeLa cell at 37 0 C in the CO 2 incubator for 24 hours and adding DMEM containing 20-500 ⁇ M ferric ammonium citrate (FAC; purchased from Sigma), the cell is incubated again at 37 0 C in the CO 2 incubator for 24 hours.
  • FAC ferric ammonium citrate
  • a pcDNA-I ⁇ B ⁇ -FTHl vector is produced by cloning the CDS of a PCR-amplified I ⁇ B ⁇ gene (Genbank Ace. No. NM_020529) into a pcDNA-FTHl vector.
  • a gene of ReIA protein Genbank Ace. No. NM 021975
  • a pEYFP-Nl vector purchased from Clonetech
  • a FRB protein and a FKBP 12 protein are interacted by having rapamycin as a mediator (Sabers, CJ., Martin, M.M., Brunn, G.J., Williams, J.M., Dumont, F. J., Wiederrecht, G. and Abraham, R.T. 1995, Isolation of a protein target of FKBP12-rapamycin complex in mammalian cells. J. Biol. Chem. 270, 815- 822).
  • a pcDNA-FKBP12-FHTl vector is produced by cloning a PCR-amplified FKBP12 gene (Genbank Ace. No.
  • a pEGFP-FRB vector is produced by cloning the CDS of a PCR-amplified FRB gene (Genbank Ace. No. NM 004958) into the pEGFP-Nl vector.
  • a mixture of the pcDNA-FKBP12-FHTl, the pBioMAGIC-mRFP-FTL and the pEGFP-FRB vectors is, according to the manner identical to the one discussed in the first embodiment, introduced into a HeLa cell.
  • the resulting cell is observed with a confocal laser microscope while being exposed to a magnetic field and in a non-magnetic-field-applied condition. From the observation, a change in green fluorescent light caused by an interaction between the FKBP12-rapamycin complex and the FRB protein is detected (Fig. 8).
  • Detection of change in protein complex by signal transduction To detect a change of protein complex caused by signal transduction in vivo, a system of TNF- ⁇ /I ⁇ B ⁇ signal transduction is selected.
  • An I ⁇ B ⁇ controls a variety of signals by forming a complex consisting of NF- ⁇ B proteins such as RelA/p65, p50, C- ReI and the like.
  • the 32nd serine and the 36th serine of an I ⁇ B ⁇ are phosphorylated by the increase of I ⁇ B ⁇ kinase (IKK) activity, and thereby causing the I ⁇ B ⁇ protein and the ⁇ TrCP protein to combine [Brown, et al., Science 267: 1485, (1995)].
  • IKK I ⁇ B ⁇ kinase
  • a protein complex forming by signal transduction By inserting the PCR-amplified I ⁇ B ⁇ gene and the CDS of a ECFP gene into a pcDNA-FTHl vector, a pcDNA-ECFP-I ⁇ B ⁇ -FTHl vector is produced. Further, a pmRFP- ⁇ TrCP vector, of which the PCR-amplified ⁇ TrCP gene is fused with a mRFP gene, is produced.
  • the EYFP-ReIA fusion protein forming a complex with the I ⁇ B ⁇ is observed to be moved by externally applied magnetic force regardless of TNF- ⁇ treating, whereas the mRFP- ⁇ TrCP fusion protein forming a complex with the phosphorylated I ⁇ B ⁇ is observed to be moved only when treated with the TNF- ⁇ . (Fig. 7)
  • a pBioMAGIC-IKK ⁇ -FTH vector is produced by cloning the CDS of a PCR-amplified IKK ⁇ gene into a FTHl expression vector and a pmRFP-I ⁇ B ⁇ vector is produced by cloning the CDS of a PCR-amplified I ⁇ B ⁇ gene into a mRFP expression vector.

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  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé qui permet de détecter les interactions entre diverses molécules d'intérêt, telles que des molécules bioactives, lequel procédé est utilisé tant pour les analyses in vitro qu'in vivo. L'invention porte sur un 'premier matériau de détection' auquel est liée une protéine magnétique qui est transloquée sous l'effet d'une force magnétique appliquée de l'extérieur, et sur un 'second matériau de détection' auquel est lié un marqueur. On peut par conséquent détecter un complexe formé par les interactions in vivo et in vitro du 'premier matériau de détection' et du 'second matériau de détection' en faisant varier la puissance de la force magnétique appliquée de l'extérieur. L'invention permet de facilement détecter et cribler une molécule cible.
PCT/KR2006/004772 2005-11-15 2006-11-14 Systeme permettant de detecter les interactions moleculaires WO2007058454A1 (fr)

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KR20050109081 2005-11-15

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2008053229A1 (fr) * 2006-11-02 2008-05-08 Iti Scotland Limited Système de reconnaissance magnétique
WO2009133202A1 (fr) * 2008-05-02 2009-11-05 Iti Scotland Limited Ensemble de marqueurs magnétiques
WO2013022313A2 (fr) * 2011-08-10 2013-02-14 Kim Tae Kook Procédé de détection quantitative et de marquage efficace d'une interaction avec une matière cible au moyen d'un transfert d'énergie et d'un changement de signal sur la base d'une présentation à haute densité d'un matériau
WO2013022299A2 (fr) * 2011-08-10 2013-02-14 Kim Tae Kook Méthode permettant d'induire un ajustement physiologique utilisant un affichage de matière haute densité
KR101433331B1 (ko) * 2007-08-17 2014-08-22 한국과학기술원 물질의 상호작용 검출 방법

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053229A1 (fr) * 2006-11-02 2008-05-08 Iti Scotland Limited Système de reconnaissance magnétique
EP2428799A1 (fr) * 2006-11-02 2012-03-14 ITI Scotland Limited Système de reconnaissance magnétique
KR101433331B1 (ko) * 2007-08-17 2014-08-22 한국과학기술원 물질의 상호작용 검출 방법
KR101547295B1 (ko) 2007-08-17 2015-08-26 한국과학기술원 물질의 상호작용 검출 방법
WO2009133202A1 (fr) * 2008-05-02 2009-11-05 Iti Scotland Limited Ensemble de marqueurs magnétiques
JP2011520101A (ja) * 2008-05-02 2011-07-14 アイティーアイ・スコットランド・リミテッド 磁性標識セット
WO2013022313A2 (fr) * 2011-08-10 2013-02-14 Kim Tae Kook Procédé de détection quantitative et de marquage efficace d'une interaction avec une matière cible au moyen d'un transfert d'énergie et d'un changement de signal sur la base d'une présentation à haute densité d'un matériau
WO2013022299A2 (fr) * 2011-08-10 2013-02-14 Kim Tae Kook Méthode permettant d'induire un ajustement physiologique utilisant un affichage de matière haute densité
WO2013022313A3 (fr) * 2011-08-10 2013-06-13 Kim Tae Kook Procédé de détection quantitative et de marquage efficace d'une interaction avec une matière cible au moyen d'un transfert d'énergie et d'un changement de signal sur la base d'une présentation à haute densité d'un matériau
WO2013022299A3 (fr) * 2011-08-10 2013-06-13 Kim Tae Kook Méthode permettant d'induire un ajustement physiologique utilisant un affichage de matière haute densité
US9759717B2 (en) 2011-08-10 2017-09-12 T&K Bioinnovation Inc. Method for quantitatively sensing and effectively marking interaction with target material by using energy transfer and signal change based on high-density display of material

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