WO1997045745A1 - PROCEDES DE DETERMINATION EX VIVO ET $i(IN VITRO) - Google Patents

PROCEDES DE DETERMINATION EX VIVO ET $i(IN VITRO) Download PDF

Info

Publication number
WO1997045745A1
WO1997045745A1 PCT/GB1997/001423 GB9701423W WO9745745A1 WO 1997045745 A1 WO1997045745 A1 WO 1997045745A1 GB 9701423 W GB9701423 W GB 9701423W WO 9745745 A1 WO9745745 A1 WO 9745745A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
proteins
vitro
labelled
label
Prior art date
Application number
PCT/GB1997/001423
Other languages
English (en)
Inventor
Michael White
Douglas Hurd
Original Assignee
Amersham Pharmacia Biotech Uk Limited
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 Amersham Pharmacia Biotech Uk Limited filed Critical Amersham Pharmacia Biotech Uk Limited
Publication of WO1997045745A1 publication Critical patent/WO1997045745A1/fr

Links

Classifications

    • 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

  • This invention is concerned with protein-protein interactions More specifically, the invention relates to improved methods for determining whether pairs of proteins are capable of interacting
  • a promoter capable of initiating transcription in vitro is present in the chimeric gene which codes for the protein of interest and such a promoter may also be present in the chimeric gene encoding the known protein
  • the promoter enables the rapid transcription and translation of the proteins in vitro
  • the in vitro promoter is upstream of the gene encoding the known protein or the protein of interest but downstream of the sequence encoding the DNA- binding domain or the transcriptional activation domain This means that the interacting proteins can be expressed in vitro independently rather than as the fusion proteins of the two-hybrid system
  • This invention aims to provide an assay system which uses the benefits of ex vivo and in vitro investigations to identify pairs of interacting proteins Such an assay system may then provide the basis for high throughput drug assays using pairs of interacting proteins
  • the present invention provides a method of investigating whether two proteins are capable of interacting with one another, which method comprises a) providing nucleic acids encoding a first protein and a second protein, b) performing an ex vivo test involving producing the first and second proteins using the nucleic acids, to indicate whether the first and second proteins may be capable of interacting with one another, c) performing a further test to indicate whether the first and second proteins are capable of interacting with one another in vitro, comprising the steps of i) producing the first and second proteins using the nucleic acids, n) labelling the first protein from i) with a first label capable of participating in a proximity detection assay, in) labelling the second protein from i) with a second label capable of participating in a proximity assay with the first label, iv) contacting the labelled first protein and the labelled second protein in vitro under conditions to allow protein-protein interactions, v) detecting an interaction between the labelled first protein and the labelled second protein by means of the proximity of the first label to the
  • the proximity of the first protein label to the second protein label brought about by an interaction between the two proteins results in the production of a detectable signal
  • a detectable signal This may be achieved by eg a scintillation proximity assay (SPA) system, in which the first protein label is a radiolabel suitable for use in SPA and the second protein label is a fluorescer comprised in a solid phase
  • the detectable signal is light energy emitted when the labelled first protein interacts with the second protein which is immobilised on the solid phase, and the radiolabel is thus brought sufficiently close to the fluorescer.
  • SPA scintillation proximity assay
  • the detectable signal may be a change in an existing signal output, eg. fluorescence.
  • Fluorescence resonance energy transfer FLC is a method which works on this principle and is described by Erickson and Cerione (12). It employs two different fluorescent molecules, a donor and an acceptor, such that when these are in sufficient proximity to one another the fluorescence of the donor molecule is absorbed by the acceptor molecule and light of a longer wavelength is emitted. Thus, when there is an interaction between two proteins each of which is labelled with one of these fluorescent molecules, a detectable signal is produced.
  • the in vitro test in the method according to the invention may be performed as a homogeneous assay without the need for a separation step after the first and second proteins have been contacted under conditions to allow protein-protein interactions.
  • the preferred format for the in vitro test in the method according to the invention is a homogeneous format.
  • the invention may involve immobilising the second protein on a solid phase.
  • the solid phase may be of any suitable material and it may take a variety of forms, for example it may be a surface or surfaces of a vessel, or particles such as beads.
  • the solid phase will comprise a suitable material impregnated with a fluorescer and preferably the solid phase will be scintillation proximity assay beads.
  • the use of a solid phase to immobilise one of the proteins in the in vitro test in the method according to the invention will provide an improved method for use in high throughput screening.
  • step b) is performed using a two-hybrid assay, although other systems known in the field, such as those of Gramatikoff (9) and Bunter & guitarist (6), could be used.
  • a particularly preferred adaptation of the two-hybrid assay for use in the method according to the invention is the system described by Yavuzer et al in WO 96/30507 and (8), as already discussed herein.
  • the Yavuzer et al system employs a promoter which allows in vitro expression of one or both of the chimeric genes which encode the proteins being tested.
  • the transfer from ex vivo to in vitro testing could be brought about by amplification of the first and second protein coding regions from the two- hybrid system, preferably by the polymerase chain reaction (PCR), using a primer which contains a promoter having activity in vitro.
  • PCR polymerase chain reaction
  • the nucleic acids thus produced are capable of being expressed in vitro.
  • first and/or second proteins may be produced in an ex vivo system eg. bacterial cells such as E.coii, and then isolated for use in the in vitro test according to the invention.
  • a solid phase When a solid phase is used in the in vitro part of the assay method according to the invention, it may need to be adapted in some manner to facilitate immobilisation of the second protein.
  • the solid phase may thus have attached to it a ligand capable of specifically binding to the second protein.
  • the second protein may be provided with an attachment label which specifically binds to the ligand on the solid phase.
  • Suitable attachment labels for the second protein and ligands for the solid phase, which labels and ligands are members of specific binding pairs, are well known in the art. Examples include biotin and streptavidin; for example the second protein may be labelled with biotin and the solid phase coated with streptavidin.
  • the second protein can be immobilised on the solid phase by a variety of methods, including any of the following:
  • the second protein includes a tag of multiple histidine residues (eg. 6), it can then be immobilised on the solid phase via a metal ion-histidine link.
  • the solid phase can be coated first in i o protein A which will enable an antibody specific for the second protein to bind via its Fc portion.
  • the second protein can then be immobilised on the surface by binding to the antibody.
  • an epitope tag recognised by the antibody may be incorporated into the second protein either by means of gene fusion or by direct attachment to the second
  • the antibody could recognise an epitope within the second protein itself.
  • Labelling of the proteins may be carried out during expression of the genes in vitro.
  • the first protein may be expressed by in vitro transcription of the gene encoding it, followed by in
  • labelling could be carried out by including the label in the growth medium, eg. in a form of a radiolabelled amino acid for the first protein label.
  • labelling of the first and/or second protein could be carried out chemically in vitro once the protein has been produced Where FRET is used, this is the preferred labelling method
  • the in vitro protein-protein interaction test can be carried out by bringing the appropriately labelled first protein into contact with the labelled second protein, in a suitable aqueous environment, and the reaction monitored for any signal produced
  • Figure 1 illustrates vectors for use in the modified two-hybrid system of Yavuzer et al (8), in which the activator gene and bait gene encode the two proteins being investigated
  • Figure 2 illustrates a preferred way of carrying out the invention, using activator and bait vectors isolated from a two-hybrid system This scheme provides a practical approach to link ex vivo gene discovery by the yeast two-hybrid system to drug development assays by SPA
  • Figure 3 illustrates a scheme for identifying interacting proteins in a library versus library screen, using an automated system
  • One particular embodiment of the method according to the invention involves in vitro transcription and translation of both the first and second proteins
  • the first protein is radiolabelled during in vitro translation with eg 35 S methionine and the second protein is labelled with biotin by including eg tRNA-biotinylated lysine in the in vitro translation reaction
  • biotin by including eg tRNA-biotinylated lysine in the in vitro translation reaction
  • Automated processes are particularly desirable, and in vitro transcription/translation is one process which is capable of being automated. If large numbers of protein-protein interactions, which have been identified in vivo eg. by the two-hybrid method, are to be analysed in vitro eg.
  • a library versus library screen using a two-hybrid assay is outlined in Figure 3
  • Library versus library two-hybrid screens involve setting up an interaction assay, using a cDNA library cloned into the activator plasmid and the same cDNA library cloned into the bait plasmid PCR is performed with oligonucleotides specific for the bait and activator plasmids to isolate sequences encoding potentially interacting proteins from the in vivo assay
  • the 5' oligonucleotides contain a promoter, eg the T7 promoter
  • the solid phase for use in the invention may be chosen to provide powerful screening techniques
  • a slide or other solid support, coated with streptavidin or other suitable ligand may be used to immobilise multiple second proteins simultaneously at spaced locations on the surface
  • a supply of first protein would be applied, and any interactions would be detected after washing, for example by means of a fluorescent label or a radiolabel
  • Such an array method may also be employed for a set of different first proteins and a single second protein, or for a set of different first proteins and a set of different second proteins
  • protein as used herein in relation to the first and second proteins is considered to include peptides, polypeptides and related molecules
  • first and second proteins may consist of a fragment of a particular protein of interest
  • yeast proteins Pho4p and Pho80p have previously been shown to interact in a two-hybrid system (7)
  • the plasmid pDM22 is a vector containing an ADH1 promoter, a Gal4 transcriptional activation domain followed by a T7 promoter, a (H ⁇ st ⁇ d ⁇ ne) 6 tag, an epitope tag, a polyhnker and an ADH1 terminator
  • the Gal4 activation domain, the T7 promoter, tags and any gene cloned into the polyhnker are expressed from a S cerevisiae ADH1 promoter
  • the plasmid contains markers and origins of replication for replication in both S cerevisiae and E coli It is a plasmid, which is commonly called the activator plasmid in the yeast two-hybrid system
  • the plasmid pDM26 is a vector containing a ADH1 promoter, the LexA DNA-binding domain (10), followed
  • the ⁇ 156 PH04 DNA (7) was amplified by PCR using suitable primers, and cloned into the EcoRI and Htnd ⁇ sites of pDM22, creating the plasmid pNWO5
  • the PHO80 DNA was amplified by PCR using suitable primers and cloned into the EcoRI and H ⁇ nd ⁇ sites of plasmid pDM26, resulting in the plasmid pDM27.
  • the interaction between Pho4p and Pho ⁇ Op was shown to be functional in an ex vivo two-hybrid test.
  • the plasmids pDM27 an pNWO5 were co-transformed into the yeast strain YM4136 which has the genotype is MATa, ura3-52, his3-200 , ade2- 101 , /ys2-801 , f ⁇ 1-901 ,leu2-3, 112, ga/80-538, (11), along with a lexo-lacZ reporter plasmid, pDM28 and a significant increase in beta-galactosidase activity was detected when pDM27 and pNW05 were transformed compared with the controls (see Figure 4).
  • Pho80p was radiolabelled, by using an in vitro T7 transcription reaction with pDM27 as the template (Amersham
  • RNA transcript was then translated using a rabbit reticulocyte in vitro translation reaction (Amersham International RPN3153) and 35 S-labelled methionine.
  • a radiolabelled protein of the predicted size of the epitope tagged Pho ⁇ Op was detected by SDS-PAGE. No product was detected in an in vitro transcription/translation reaction containing no template DNA (see Figure 5).
  • the Pho4p protein was also produced in an in vitro transcription/translation reaction.
  • pNWO5 was used as a template in the in vitro transcription reaction, producing PH04 mRNA.
  • the Pho4p transcript was translated in an in vitro translation reaction including tRNA-biotinylated lysine (ECL in vitro translation system, RPN 2197 , Amersham).
  • ECL in vitro translation system RPN 2197
  • the product was detected on a Western blot using streptavidin-HRP (Amersham) and Enhanced Chemiluminescence (ECL) .
  • a protein of approximately 29 kD was detected on a SDS-PAGE gel. This is the predicted size of the T7 epitope tagged-Pho4p expressed from pNW05.
  • the 29kD protein was incubated with streptavidin-coated SPA beads (Amersham) at 4°C.
  • the T7 epitope tagged Pho4p was bound to the SPA beads. Attachment of the 29kD protein was confirmed by precipitating the SPA beads for one minute in a microfuge , and washing in 1 ml of PBS The SPA beads were resuspended in SDS PAGE loading buffer, and boiled for 5 minutes to re-elute the Pho4p from the SPA beads The eluted protein was then run on SDS PAGE, along with the in vitro transcribed and translated T7 epitope tagged Pho4p protein Also run was a control in which there was no DNA template in the in vitro transcription/translation reaction The gel was Western blotted, probed with an antibody to the T7 epitope and detected using ECL (Amersham) The T7 epitope tagged Pho4p was seen to be present on the SPA beads (see Figure 7) i o
  • Figure 4 Illustrates the results of a 2 hybrid assay for the interacting proteins Pho4p and Pho ⁇ Op
  • the following plasmids were transformed into the yeast strain YM4136 and the Beta galactosidase i s activity assayed by a method based on Harshman et al Column 1 , pNW05, pDM27 and pDM28, Column 2, pDM22, pDM27 and pDM28, Column 3, pNW05, pDM26 and pDM2 ⁇ , Column 4, pDM22, pDM26 and pDM28, Column 5, pNW05 and pDM27, Column 6, pDM28
  • Figure 5 shows 35 S methionine labelled protein produced by 0 in vitro transcription/ translation reactions (Amersham, RPN 2197) programmed with Lane 1 , no plasmid, Lane 2, pNW05, Lane 3, pDM27, Lane 4, pCITE-Beta galactosidase (positive control plasmid included in RPN 2197), Lane 5, pDM27 The reactions were separated on a 15% SDS-PAGE gel and the gel exposed to Hyperfilm MP for 3 days
  • Figure 6 shows a Western blot of biotinylated labelled protein produced by in vitro transcription and then translation reactions, in the presence of biotinylated tRNA-lysine (Amersham, RPN 2199)
  • the transcription reactions were programmed with Lane 1 , pDM27, Lane 2, pNW05, Lane 3 no DNA
  • the reactions were separated on a 15% SDS-
  • Figure 7 shows a Western blot of biotinylated labelled protein produced by in vitro transcription/translation reactions programmed with: Lanes 1 and 2, no DNA; Lanes 3 and 4, pNW05.
  • the reactions in Lanes 2 and 4 were first bound to streptavidin coated SPA beads before being eluted from the SPA beads by boiling in SDS-PAGE loading buffer for 5 minutes prior to loading onto the gel.
  • the reactions were separated on a 15% SDS-PAGE gel, Western blotted and the blot probed with a 1/1000 dilution of HRP labelled strepatavidin, followed by detection using ECL reagents (Amersham).
  • the construction of the plasmids was by standard procedures (15) Purification of DNA was carried out using Qiagen columns Enzymes were obtained from Amersham Oligonucleotides were synthesised by Genosys (Cambridge, UK) The bacterial host used was E coli strain pMosS/ue (Amersham) (endA1 hsdR1 ' 7(r M2 m M2 * )supE44th ⁇ -1 recA1 gyrA96 relA1 /ac(7 r ' pro/r ⁇ + /ac/ q Z ⁇ M 15 Tn10(Tc R ) or NM554 (F araD139 ⁇ (ara- leu)7696 ga!E15 galKW ⁇ (lac)X74 rpsL (St ⁇ hsdR2 (r k nV) mcrA mcrB1 recA13
  • ATF2 was cloned into the bait plasmid pDM26
  • the plasmid pDM26 has been described previously (WO 96/30507) and expresses in yeast a LexA DNA binding domain
  • a kanamycin resistance gene was cloned into pDM26 by excising the kanamycin resistance gene from pUC4K (Pharmacia) with Pst ⁇ The fragment was inserted between the ⁇ gll and Aatll sites of pDM26 The resulting plasmid was called pDM41
  • the ATF2 gene (a plasmid containing ATF2 was a gift from Nic Jones, ICRF, Lincolns Inn Field, London, UK) was then cloned into pDM41 (-), at a position downstream of the LexA, in a two step process Firstly, oligonucleotides (sequence AATTCCAGCTGG and GATCCCAGCTGG.
  • the PvuW site is underlined were synthesized, annealed and cloned between the EcoRI and SamHI sites of pDM41 (-) to create pSD7
  • the A TF2 gene was excised from the ATF2 plasmid using Sa/I and C/al
  • the fragment was treated with T4 polymerase to create blunt ends and inserted into the PvuW site of pSD7, to produce the plasmid pSD12
  • the plasmid pSD12 therefore contained the first chimeric gene encoding, in this example, the LexA DNA binding domain fused to ATF2
  • the construction of the plasmid pDM27 which is a differential expression vector containing the PH04N ⁇ 156 inserted into the multicloning site has been described previously (WO 96/30507).
  • the plasmid containing cJun was cloned from a human brain cDNA library which had been inserted into the activator plasmid pNW17 .
  • the activator plasmid is based on pDM22 (WO 96/30507).
  • the plasmid pDM22 expresses the Gal4p transcriptional activation domain. This plasmid was modified by the addition of an F1 origin of replication.
  • the Hind ⁇ sites at the junction of the ADH1 promoter with the GAL4 activation domain and at the 5' end of the ADH1 terminator were removed by excising the Hind ⁇ fragment, treating the fragment with T4 DNA polymerase and recloning the blunt-ended Hind ⁇ fragment back into the Hind ⁇ site of the activator plasmid to create pNW17.
  • This plasmid therefore contained a unique Hind ⁇ site in the polyhnker pNW17.
  • a cDNA library was cloned into pNW17 as described below.
  • the cDNA library (constructed using the Novagen directional cDNA construction kit) was cloned between the EcoRI and HindlW sites of pNW17.
  • the two hybrid screen was carried out using a bait plasmid which expresses a LexA DNA binding domain-ATF2 hybrid fusion protein.
  • the reporters used were a lexAo-HIS3 and a lexo-lacZ. Extraction of activator plasmids from His+ lacZ+ yeast resulted in the identification of an activator plasmid which contained the complete cJun gene.
  • This plasmid therefore expresses in yeast a Gal4 activation domain-cJun fusion protein, and was named pACT#jun.
  • the construction of the plasmid pNW05 which expresses a Gal4p activation doma ⁇ n-Pho4N ⁇ l56 protein has been described elsewhere (WO 96/30507)
  • the Pho4N ⁇ l 56 protein is not expected to interact with ATF2 protein
  • Beta galactosidase assays were carried out as described previously (WO 96/30507) The beta-galactosidase activity was significantly higher in the pACT#jun, pSD12 and pDM28 transformant compared to the yeast strains containing pNW05 (PH04 ⁇ 156 containing activator), pSD12 and pDM28 or ⁇ ACT#jun, pDM41(-) (the empty bait) and pDM2 ⁇ (see table 2) Beta galactosidase units were calculated using the following formula
  • Beta-galactosidase units 1000 x OD at 420/(t x V x OD at 600)
  • the beads were harvested as described above, washed three times with 0.7 ml of binding buffer before being resuspended in 0.7 ml of binding buffer. The suspension was then counted in a scintillation counter. The results are shown in figure 8.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • 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)
  • Peptides Or Proteins (AREA)

Abstract

Procédé permettant d'identifier des paires de protéines en interaction et consistant à rechercher par analyse les interactions possibles entre les protéines dans un système ex vivo, à sélectionner des paires de protéines présentant une interaction apparente et à effectuer une autre analyse dans un système in vitro dans lequel les protéines sont marquées, de façon à produire un signal détectable résultant de la proximité des marquages quand les protéines entrent en interaction.
PCT/GB1997/001423 1996-05-24 1997-05-23 PROCEDES DE DETERMINATION EX VIVO ET $i(IN VITRO) WO1997045745A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP96303749 1996-05-24
EP96303749.4 1996-05-24

Publications (1)

Publication Number Publication Date
WO1997045745A1 true WO1997045745A1 (fr) 1997-12-04

Family

ID=8224953

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/001423 WO1997045745A1 (fr) 1996-05-24 1997-05-23 PROCEDES DE DETERMINATION EX VIVO ET $i(IN VITRO)

Country Status (1)

Country Link
WO (1) WO1997045745A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001027633A (ja) * 1999-05-07 2001-01-30 Inst Of Physical & Chemical Res 相互作用するタンパク質の検出方法
US7335866B2 (en) 2002-10-28 2008-02-26 Carl Zeiss Microimaging Gmbh Method for improving depth discrimination in optical reproduction systems
WO2017171606A1 (fr) * 2016-03-29 2017-10-05 Ridgeview Instruments Ab Procédé de détection de molécules se trouvant à proximité immédiate

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4568649A (en) * 1983-02-22 1986-02-04 Immunex Corporation Immediate ligand detection assay
EP0234799A2 (fr) * 1986-02-10 1987-09-02 AMERSHAM INTERNATIONAL plc Procédé pour la détection ou l'isolement de proteine
US5283173A (en) * 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO1995034664A1 (fr) * 1994-06-16 1995-12-21 Genetics Institute, Inc. Procede de detection d'interactions de ligands
GB2291708A (en) * 1994-07-22 1996-01-31 Zeneca Ltd Process for screening a library of compounds released from a solid phase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4568649A (en) * 1983-02-22 1986-02-04 Immunex Corporation Immediate ligand detection assay
EP0234799A2 (fr) * 1986-02-10 1987-09-02 AMERSHAM INTERNATIONAL plc Procédé pour la détection ou l'isolement de proteine
US5283173A (en) * 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO1995034664A1 (fr) * 1994-06-16 1995-12-21 Genetics Institute, Inc. Procede de detection d'interactions de ligands
GB2291708A (en) * 1994-07-22 1996-01-31 Zeneca Ltd Process for screening a library of compounds released from a solid phase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PHIZICKY ET AL.: "Protein-protein interactions: Methods for detection and analysis", MICROBIOLOGICAL REVIEWS, vol. 59, no. 1, March 1995 (1995-03-01), WASHINGTON, DC, pages 94 - 123, XP000196498 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001027633A (ja) * 1999-05-07 2001-01-30 Inst Of Physical & Chemical Res 相互作用するタンパク質の検出方法
EP1182458A1 (fr) * 1999-05-07 2002-02-27 Riken Methode de detection de proteines en interaction mutuelle
EP1182458A4 (fr) * 1999-05-07 2005-02-16 Riken Methode de detection de proteines en interaction mutuelle
US7122382B1 (en) 1999-05-07 2006-10-17 Riken Method for detecting proteins under mutual interaction
JP4493125B2 (ja) * 1999-05-07 2010-06-30 独立行政法人理化学研究所 相互作用するタンパク質の検出方法
US7335866B2 (en) 2002-10-28 2008-02-26 Carl Zeiss Microimaging Gmbh Method for improving depth discrimination in optical reproduction systems
WO2017171606A1 (fr) * 2016-03-29 2017-10-05 Ridgeview Instruments Ab Procédé de détection de molécules se trouvant à proximité immédiate

Similar Documents

Publication Publication Date Title
JP5415264B2 (ja) 検出可能な核酸タグ
US5846722A (en) System to detect small molecule/peptide interaction
Pollock et al. A sensitive method for the determination of protein-DNA binding specificities
US6562576B2 (en) Yeast two-hybrid system and use thereof
US20080214408A1 (en) In situ assembly of protein microarrays
EP0752477A2 (fr) Vecteurs d'expression pour la production de récepteurs stéroides, chimères de ces récepteurs, tests de dépistage pour ces récepteurs et tests cliniques utilisants les récepteurs synthétisés ainsi que leurs vecteurs
EP1182458B1 (fr) Methode de detection de proteines en interaction mutuelle
EP1937848B1 (fr) Assemblage in situ de puces à proteines
WO1997045745A1 (fr) PROCEDES DE DETERMINATION EX VIVO ET $i(IN VITRO)
JP2003524371A (ja) アッセイ方法
US20080248958A1 (en) System for pulling out regulatory elements in vitro
WO2008140538A1 (fr) Ecran d'affichage d'adn pour un produit d'expression avec des propriétés de liaison désirées
US7932030B2 (en) System for pulling out regulatory elements using yeast
JP4172332B2 (ja) 核内受容体リガンド検出剤、組換えタンパク質、発現用遺伝子、組換えベクター、形質転換体、核内受容体リガンド検出方法、及び、核内受容体リガンド検出キット
EP1513935A1 (fr) Vecteurs destines a l'expression de proteines biotinylees dans des cellules mammaliennes et leur utilisation pour identifier les interactions proteines - acides nucleiques in vivo
US20050239078A1 (en) Sequence tag microarray and method for detection of multiple proteins through DNA methods
JP5390221B2 (ja) 被検物質を検出するためのポリヌクレオチド、前記ポリヌクレオチドを含むベクターおよびそれらを用いた検出方法
WO2001083808A2 (fr) Systeme de produit de recombinaison d'acide nucleique et procede utilisant celui-ci pour identifier des interactions entre des proteines
Jing Identification of the Na/K-ATPase interacting proteins
US20030100022A1 (en) Nucleic acid construct system and method utilizing same useful for identifying protein-protein interactions
WO2003035911A1 (fr) Identification de clone par detection directe de proteines de liaison aux acides nucleiques provenant de systemes d'expression de cellules de vertebres et applications de ce procede

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA GB JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97541837

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase