WO2000014271A1 - PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO) - Google Patents

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

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Publication number
WO2000014271A1
WO2000014271A1 PCT/US1999/020207 US9920207W WO0014271A1 WO 2000014271 A1 WO2000014271 A1 WO 2000014271A1 US 9920207 W US9920207 W US 9920207W WO 0014271 A1 WO0014271 A1 WO 0014271A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
complexed
acceptor fluorophore
cell
luciferase
Prior art date
Application number
PCT/US1999/020207
Other languages
English (en)
Inventor
Aladar A. Szalay
Yubao Wang
Gefu Wang-Pruski
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
Priority to AU58056/99A priority Critical patent/AU752675B2/en
Priority to JP2000569011A priority patent/JP2002524087A/ja
Priority to CA002341314A priority patent/CA2341314A1/fr
Priority to EP99945460A priority patent/EP1109931A4/fr
Publication of WO2000014271A1 publication Critical patent/WO2000014271A1/fr

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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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  • Biotechnology (AREA)
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  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (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.
PCT/US1999/020207 1998-09-03 1999-09-02 PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO) WO2000014271A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU58056/99A AU752675B2 (en) 1998-09-03 1999-09-02 Method for studying protein interactions (in vivo)
JP2000569011A JP2002524087A (ja) 1998-09-03 1999-09-02 インビボにおいてタンパク質相互作用を調べる方法
CA002341314A CA2341314A1 (fr) 1998-09-03 1999-09-02 Procede d'etude d'interactions de proteines in vivo
EP99945460A EP1109931A4 (fr) 1998-09-03 1999-09-02 PROCEDE D'ETUDE D'INTERACTIONS DE PROTEINES $i(IN VIVO)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9906898P 1998-09-03 1998-09-03
US60/099,068 1998-09-03
US13583599P 1999-05-24 1999-05-24
US60/135,835 1999-05-24

Publications (1)

Publication Number Publication Date
WO2000014271A1 true WO2000014271A1 (fr) 2000-03-16

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

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1533619A2 (fr) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique.
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
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
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
US8647887B2 (en) 2009-01-29 2014-02-11 Commonwealth Scientific And Industrial Research Organisation Measuring G protein coupled receptor activation
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
US10415960B2 (en) 2015-04-06 2019-09-17 Worldvu Satellites Limited Elevation angle estimating system and method for user terminal placement
US11579149B2 (en) 2017-11-01 2023-02-14 Queen's University At Kingston Hippo pathway bioluminescent biosensor

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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
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
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
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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US4318707A (en) * 1978-11-24 1982-03-09 Syva Company Macromolecular fluorescent quencher particle in specific receptor assays
US5683888A (en) * 1989-07-22 1997-11-04 University Of Wales College Of Medicine Modified bioluminescent proteins and their use
US5418155A (en) * 1989-12-29 1995-05-23 University Of Georgia Research Foundation, Inc. Isolated Renilla luciferase and method of use thereof
US5866348A (en) * 1993-02-10 1999-02-02 Parkard Instrument Company, Inc. Bioluminescence measurement system
US5491084A (en) * 1993-09-10 1996-02-13 The Trustees Of Columbia University In The City Of New York Uses of green-fluorescent protein
US5811238A (en) * 1994-02-17 1998-09-22 Affymax Technologies N.V. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5976796A (en) * 1996-10-04 1999-11-02 Loma Linda University Construction and expression of renilla luciferase and green fluorescent protein fusion genes
US5891646A (en) * 1997-06-05 1999-04-06 Duke University Methods of assaying receptor activity and constructs useful in such methods

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
EP2053409A1 (fr) 2003-11-20 2009-04-29 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique
EP1533619A2 (fr) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Marqueurs spécifiques du syndrome métabolique.
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
US9605069B2 (en) 2008-02-29 2017-03-28 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM a protein and uses thereof
US8647887B2 (en) 2009-01-29 2014-02-11 Commonwealth Scientific And Industrial Research Organisation Measuring G protein coupled receptor activation
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
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
WO2011131626A1 (fr) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 code pour l'activité alkylglycérol monooxygénase dépendante de la tétrahydrobioptérine
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US9365643B2 (en) 2012-01-27 2016-06-14 AbbVie Deutschland GmbH & Co. KG Antibodies that bind to repulsive guidance molecule A (RGMA)
US10106602B2 (en) 2012-01-27 2018-10-23 AbbVie Deutschland GmbH & Co. KG Isolated monoclonal anti-repulsive guidance molecule A antibodies and uses thereof
US10415960B2 (en) 2015-04-06 2019-09-17 Worldvu Satellites Limited Elevation angle estimating system and method for user terminal placement
US11579149B2 (en) 2017-11-01 2023-02-14 Queen's University At Kingston Hippo pathway bioluminescent biosensor

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Publication number Publication date
JP2002524087A (ja) 2002-08-06
CN1160470C (zh) 2004-08-04
AU752675B2 (en) 2002-09-26
CN1323353A (zh) 2001-11-21
AU5805699A (en) 2000-03-27
EP1109931A4 (fr) 2004-12-15
EP1109931A1 (fr) 2001-06-27
CA2341314A1 (fr) 2000-03-16

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