EP1109931A1 - PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO) - Google Patents

PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO)

Info

Publication number
EP1109931A1
EP1109931A1 EP99945460A EP99945460A EP1109931A1 EP 1109931 A1 EP1109931 A1 EP 1109931A1 EP 99945460 A EP99945460 A EP 99945460A EP 99945460 A EP99945460 A EP 99945460A EP 1109931 A1 EP1109931 A1 EP 1109931A1
Authority
EP
European Patent Office
Prior art keywords
protein
complexed
acceptor fluorophore
cell
luciferase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99945460A
Other languages
German (de)
English (en)
Other versions
EP1109931A4 (fr
Inventor
Aladar A. Szalay
Yubao Wang
Gefu Wang-Pruski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Loma Linda University
Original Assignee
Loma Linda University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Loma Linda University filed Critical Loma Linda University
Publication of EP1109931A1 publication Critical patent/EP1109931A1/fr
Publication of EP1109931A4 publication Critical patent/EP1109931A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the method could be used with a wide variety of proteins and in a wide variety of living cells. Also preferably, the method could be used to determine the interactions between molecules other than proteins.
  • a method for determining whether a first protein interacts with a second protein within a living cell comprises providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore within the cell.
  • the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first protein is in proximity to the second protein.
  • the complexed first protein and the complexed second protein are allowed to come into proximity to each other within the cell.
  • any fluorescence from the acceptor fluorophore is detected. Fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase to acceptor fluorophore the indicates that the first protein has interacted with the second protein.
  • providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to the living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore.
  • the cell which is provided with the first protein complexed to a donor luciferase and the cell which is provided with the second protein complexed to an acceptor fluorophore are mammalian cells.
  • the donor luciferase provided is Renilla luciferase.
  • the acceptor fluorophore provided is an Aequorea green fluorescent protein.
  • the detection of acceptor fluorophore fluorescence is performed using spectrofluorometery.
  • the present invention includes a method for determining whether a first protein interacts with a second protein in a living cell using luminescent resonance energy transfer (LRET).
  • LRET luminescent resonance energy transfer results from the transfer of excited state energy from a donor luciferase to an acceptor fluorophore.
  • LRET luminescent resonance energy transfer
  • the efficiency of luminescence resonance energy transfer is dependent on the distance separating the donor luciferase and the acceptor fluorophore, among other variables. Generally, significant energy transfers occur only where the donor luciferase and acceptor fluorophore are less than about 80 A of each other.
  • the present invention utilizes luminescence resonance energy transfer to determine whether an interaction takes place between a first protein and a second protein in a living cell. This is accomplished by complexing a first protein to the donor luciferase and complexing the second protein to the acceptor fluorophore and placing the complexed first protein and the complexed second protein in the cell under conditions suitable for an interaction between the first protein and the second protein to take place. If the first protein interacts with the second protein, the donor luciferase will come close enough to the acceptor fluorophore for luminescence resonance energy transfer to take place and the acceptor fluorophore will fluoresce.
  • this method allows for the detection of interaction between the first protein and the second protein even though the interaction cannot be detected by optical methods such as conventional microscopy.
  • the specific labeling of the proteins in living cells can be achieved through genetic engineering methods where the introduction of fluorescent dyes into living cells is very difficult. Further, fluorescent dyes photobleach quickly while light emission of a luciferase such as Renilla luciferase originates from an enzymatic reaction that is relatively stable if substrate and oxygen are supplemented.
  • complexing a first protein to the donor luciferase refers to joining the donor luciferase to the first protein in a manner that the donor luciferase and the first protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein.
  • complexing a second protein to the acceptor fluorophore refers to joining the acceptor fluorophore to the second protein in a manner that the acceptor fluorophore and the second protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein.
  • Such complexing can be done, for example, by genetically engineering the cell to produce a fusion protein containing the donor luciferase and first protein, and the acceptor fluorophore and the second protein.
  • the present invention uses Renilla luciferase as the donor luciferase and "humanized" Aequorea green fluorescent protein ('humanized' GFP) as the acceptor fluorophore.
  • Renilla luciferase is a 34 kDa enzyme purified from Renilla reniformis. The enzyme catalyzes the oxidative decarboxylation of coelenterazine in the presence of oxygen to produce blue light with an emission wavelength maximum of 471 nm.
  • Renilla luciferase was used as the donor luciferase because it requires an exogenous substrate rather than exogenous light for excitation. This, advantageously, eliminates background noise from an exogenous light source and from autofluorescence, and allows easy and accurate quantitative determination of light production.
  • 'Humanized' GFP is a 27 kDa protein fluorophore that has an excitation maximum at 480 nm. It has a single amino acid difference from wild-type Aequorea green fluorescent protein. 'Humanized' GFP was chosen as the acceptor fluorophore because its excitation spectrum overlaps with the emission spectra of Renilla luciferase. Additionally, emissions from 'humanized' GFP can be visualized in living cells. Further, 'humanized'
  • GFP is expressed well in the mammalian cells transfected with 'humanized' GFP cDNA that were used to demonstrate this method.
  • IGFBP 6 insulin-like growth factor binding protein 6
  • IGF- II insulin-like growth factor II
  • the Renilla luciferase cDNA was fused to IGFBP 6 cDNA and 'humanized' GFP cDNA was fused to IGF-II cDNA.
  • Living cells were transfected with the fused cDNAs and the fusion proteins were expressed. Cell extracts were produced and mixed.
  • the substrate for the Renilla luciferase moiety of the fused Renilla luciferase-IGFPB 6 protein was added. Finally, fluorescence from the 'humanized' GFP moiety of the fused 'humanized' GFP-IGF-II protein was detected. Demonstration one method according to the present invention will now be described in greater detail.
  • IGFBP-6 cDNA SEQ ID NO: l, GenBank accession number M69054, encoded IGFBP-6, SEQ ID NO:2, which was used as the first protein.
  • Renilla luciferase cDNA SEQ ID NO:3, GenBank accession number M63501, encoded Renilla luciferase, SEQ ID NO:4, which was used as the donor luciferase.
  • Insulin cDNA SEQ ID NO:9, accession number AH002844, encoded insulin, SEQ ID NO: 10.
  • Insulin, fused to 'humanized' GFP was used as a control protein because insulin is homologous to IGF-II, but it does not bind to IGFBP-6.
  • the cDNA of prepro-IGF-II carried on an EcoRI fragment was cloned into pBluescript KS (+) II vector.
  • the insert was sequenced using T7 and T3 primers and confirmed to contain the known cDNA sequence of prepro-IGF-II.
  • the 5' end of the IGF-II precursor was connected to the T7 promoter in the pBluescript KS (+) II vector.
  • An IGF-II 3' primer was designed to generate a Notice of Allowance restriction site, to remove the D and E domains of prepro-IGF-II, and to maintain the Notice of Allowance fragment of the 'humanized' GFP in frame with the open reading frame of IGF-II.
  • the IGF-II fragment was amplified with PCR using the T7 promoter primer and the IGF-II 3' primer.
  • the PCR-amplified IGF-II fragment was digested by EcoRI and Not I and cloned into pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, US) producing pCDNA-IGF-II.
  • the Notice of Allowance fragment of the 'humanized' GFP was inserted into the Not I site of pCDNA-IGF-II producing pC-IGF-II-GFP.
  • the cDNA for precursor of insulin which contained a signal peptide the B, C and A domains, was modified in a manner corresponding to the IGF-II fragment, above.
  • the 'humanized' GFP cDNA was then linked to the 3' end of the modified insulin cDNA to produce pC-INS-GFP.
  • IGFBP 6 cDNA was amplified by PCR from a plasmid named
  • Rat-tagged human IGFBP6 Rat-tagged human IGFBP6.
  • the stop codon of IGFBP 6 was removed and the open reading frame of IGFBP 6 was in frame with Renilla luciferase cDNA from pCEP4-RUC (Mayerhofer R, Langridge WHR, Cormier MG and Szalay AA. Expression of recombinant Renilla luciferase in trans genie plants results in high levels of light emission. The Plant Journal 1995 ;7; 1031-8).
  • the linking of the Renilla luciferase cDNA to the 3' end of modified IGFBP 6 cDNA produced pC-IGFBP 6-RUC.
  • COS-7 cells African green monkey kidney cell, American Type Culture Collection CRL 1651
  • DMEM Dulbecco's Modified Eagle Medium
  • streptomycin 100 mg/ml antibiotic antimycotic solution containing a final concentration of penicillin 100 unit/ml, streptomycin 100 mg/ml and amphotericin B 250 ng/ml (Sigma-Aldrich Co., St. Louis, MO, US) in 5% CO 2 .
  • Groups of 1x10° of these cells were plated the day before transfection and were approximately 50% to 60% confluent at the time of transfection. Forty mg of each plasmid fusion DNA were precipitated and resuspended into
  • fusion proteins IGF-II-GFP and IGFBP 6-RUC having the expected molecular weights of about 36 kDa and 56 kDa, respectively, were detected using immunoblot analysis. This confirmed the presence of both fusion proteins in the transiently transfected cells.
  • cell extracts from these transiently transfected cells were used to carry out a protein binding assay based on energy transfer between the Renilla luciferase and 'humanized' GFP moieties of the fusion proteins.
  • the COS cells were washed twice with PBS and harvested using a cell scraper in luciferase assay buffer containing 0.5 M NaCl, 1 mM EDTA and 0.1 M potassium phosphate at a pH 7.5.
  • the harvested cells were sonicated 3 times for 10 seconds with an interval of 10 seconds using a Fisher Model 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, US) to produce cell extracts.
  • the cell extracts containing IGF-II-GFP and IGFBP 6-RUC were mixed and 0.1 ⁇ g of coelenterazine was immediately added.
  • Spectrofluorometry was performed using a SPEX FluoroMax ® (Instruments S.A., Inc., Edison, NJ). The spectrum showed a single emission peak at 471 nm, which corresponds to the known emission of Renilla luciferase.
  • the spectrofluorometry of the cell extracts was carried out at a longer time, but the spectral pattern did not change over time.
  • Control cell extract mixtures from cells transfected with pC-INS-GFP and pC-IGFBP 6-RUC were made similarly and their spectra traced.
  • the traces showed only one peak at 471 nm, which corresponds to the emission peak of Renilla luciferase.
  • the spectral pattern did not change over time.
  • protein-protein interactions were also detected by the detection of LRET using corresponding methods in E. coli cells and mammalian cells which were co-transformed.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé servant à déterminer si une première protéine interagit avec une deuxième protéine dans une cellule vivante. Le procédé comporte l'étape consistant à faire en sorte que la première protéine forme un complexe avec une luciférase donneur, et que la deuxième protéine forme un complexe avec un fluorophore accepteur dans la cellule. La première protéine formant un complexe et la deuxième protéine formant un complexe sont amenées à proximité l'une de l'autre dans la cellule. Ensuite, on détecte toute fluorescence provenant du fluorophore accepteur, qui résulte d'un transfert d'énergie par résonance de luminescence provenant de la luciférase donneur, la fluorescence provenant du fluorophore accepteur indiquant que la première protéine a interagi avec la deuxième protéine.
EP99945460A 1998-09-03 1999-09-02 PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO) Withdrawn EP1109931A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US9906898P 1998-09-03 1998-09-03
US99068P 1998-09-03
US13583599P 1999-05-24 1999-05-24
US135835P 1999-05-24
PCT/US1999/020207 WO2000014271A1 (fr) 1998-09-03 1999-09-02 PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO)

Publications (2)

Publication Number Publication Date
EP1109931A1 true EP1109931A1 (fr) 2001-06-27
EP1109931A4 EP1109931A4 (fr) 2004-12-15

Family

ID=26795497

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99945460A Withdrawn EP1109931A4 (fr) 1998-09-03 1999-09-02 PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO)

Country Status (6)

Country Link
EP (1) EP1109931A4 (fr)
JP (1) JP2002524087A (fr)
CN (1) CN1160470C (fr)
AU (1) AU752675B2 (fr)
CA (1) CA2341314A1 (fr)
WO (1) WO2000014271A1 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003280420A1 (en) 2002-06-26 2004-01-19 Yale University Modulators and modulation of the interacton between rgm and neogenin
DE602004020855D1 (de) 2003-11-20 2009-06-10 Hoffmann La Roche Spezifische Marker für Stoffwechselssyndrome
CN1920021B (zh) * 2005-08-24 2010-05-05 中国医学科学院基础医学研究所 胰岛素样生长因子结合蛋白-6介导的有活性胰岛素样生长因子-ⅱ的制备方法
ES2542501T3 (es) 2005-09-30 2015-08-06 Abbvie Deutschland Gmbh & Co Kg Dominios de unión de proteínas de la familia de proteínas de moléculas de orientación repulsiva (RGM) y fragmentos funcionales de las mismas, así como su uso
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
CN102439444B (zh) 2009-01-29 2014-10-22 联邦科学技术研究组织 测量g蛋白偶联受体激活
CN101620233B (zh) * 2009-05-27 2012-10-31 华中科技大学 一种蛋白质相互作用的检测方法
ES2562832T3 (es) 2009-12-08 2016-03-08 Abbvie Deutschland Gmbh & Co Kg Anticuerpos monoclonales contra la proteína RGM para su uso en el tratamiento de la degeneración de la capa de fibra nerviosa de la retina
WO2011083147A1 (fr) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Inhibition de wave1 dans l'intervention médicale de maladies inflammatoires et/ou d'infections causées par un pathogène
EP2561068A1 (fr) 2010-04-19 2013-02-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
BR112014018592B1 (pt) 2012-01-27 2022-03-15 Abbvie Inc. Anticorpo monoclonal isolado que se liga à molécula de orientação repulsiva a (rgma)
CN102798717B (zh) * 2012-06-15 2014-11-26 杭州师范大学 一种o6-甲基鸟嘌呤-dna甲基转移酶活性检测方法
CN103616502B (zh) * 2013-09-12 2016-05-25 西北农林科技大学 基于细菌荧光素酶bret技术检测蛋白质相互作用的方法
US10415960B2 (en) 2015-04-06 2019-09-17 Worldvu Satellites Limited Elevation angle estimating system and method for user terminal placement
CA3022981A1 (fr) 2017-11-01 2019-05-01 Queen's University At Kingston Biocapteur bioluminescent de parcours hippo
CN110794129B (zh) * 2018-08-01 2020-12-01 清华大学 细胞内检测生物分子间相互作用及其调控因子的方法与所用试剂

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EP0137515A2 (fr) * 1983-10-13 1985-04-17 The University Of Georgia Research Foundation, Inc. Immunoessais bioluminescents
WO1999066324A2 (fr) * 1998-06-16 1999-12-23 Biosignal Packard Inc. Systeme de transfert d'energie de resonance par bioluminescence et utilisation dudit systeme

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Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0137515A2 (fr) * 1983-10-13 1985-04-17 The University Of Georgia Research Foundation, Inc. Immunoessais bioluminescents
WO1999066324A2 (fr) * 1998-06-16 1999-12-23 Biosignal Packard Inc. Systeme de transfert d'energie de resonance par bioluminescence et utilisation dudit systeme

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WANG Y ET AL: "Chemiluminescence energy transfer to study protein-protein interaction in living cells" BIOLUMINESCENCE AND CHEMILUMINESCENCE FOR THE 21ST CENTRUY - PROCEEDINGS OF THE 10TH INTERNATIONAL SYMPOSIUM ON BIOLUMINESCENCE AND CHEMILUMINESCENCE, 1999, pages 475-478, XP008036438 *

Also Published As

Publication number Publication date
JP2002524087A (ja) 2002-08-06
WO2000014271A1 (fr) 2000-03-16
CN1160470C (zh) 2004-08-04
AU5805699A (en) 2000-03-27
AU752675B2 (en) 2002-09-26
EP1109931A4 (fr) 2004-12-15
CN1323353A (zh) 2001-11-21
CA2341314A1 (fr) 2000-03-16

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