CA2301633A1 - Method and kit for identifying interactions between proteins or peptides - Google Patents
Method and kit for identifying interactions between proteins or peptides Download PDFInfo
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- CA2301633A1 CA2301633A1 CA002301633A CA2301633A CA2301633A1 CA 2301633 A1 CA2301633 A1 CA 2301633A1 CA 002301633 A CA002301633 A CA 002301633A CA 2301633 A CA2301633 A CA 2301633A CA 2301633 A1 CA2301633 A1 CA 2301633A1
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Abstract
The invention relates to a method and kit for identifying interactions between proteins or peptides by means of fluorescence resonance energy transfer.
Description
DESCRTPTION
?iLTROD ARD ICIT F08 IDENTIFYING 119TERAGTIONS BE1'ilEEl~ PROTEINS OR PEPTIDES
The present invention relates to a method and to a kit for the identification or determination of interactions between prot~ias or peptides by means of fluorescence-resonance energy transfer.
The identificatiott_and analysis of interactions bets~reen different proteins or peptides or fragments thereof represents as important problem of biopiedical research and biotechnology. At the end of the 1980's a system W88 coo- .
sequently developed, which acquired great research signif~.cance under the name "Yeast Two-Nyhrid-System" (Fields et ai.,.Natnre, vol.. 340, pp z45~247, 1489). Ti~.ts system is based on the discovery that cellular transcription activators,. such as e.g. GAL4 or lexA from yeast could be deCOmposed in Lw0 f independent fvmtctloa domains. Both domains are normally a component .af a protein in the nucleus of the yeast cell, which binds to.specific actipatiag sequences~~of diffazeat target genes and regulates the transcription thereof.
Ons domain,~the~.DNA~~binding domain (8D), specifically binds to.a particular DHA target seqaeace (upstream activating seqaeace) in the vicinity of the target gene promoter. The oilier domain, the activatins domaf.u (AD), increases the transcription rate of the target gene by interaction pith the transcription initiation complex, which is bound to the protater of the target gene. In the "Yeast Two-Hpbrid System" this structure is utilized by transcription factors in modified form. The DNA binding domain (BD) of GAL4 or lesA is there expressed as a fuaioa protein with a "baiting protein. or peptide" in yeast cells. This fusion protein also has a nucleus location shgnai through which it is transported into the yeast nucleus. The.baiting-fusi.on protein is bound there to a target sequence (UAS), which in the yeast strain used is located in the viciaf.ty of prvmaters of two reporter genes (e. g. anxotrophic i~aarker (HIS3) and enzy~tatic mar$er (lacZ)). This leads to s'conStellatioa in which the baiting protein or peptide is exposed ~in direct. spatial proximity to the reporter gene promoter. Additionally a second fusion protein is expressed in the same yeast.cell. Zt Comprises the activating domain (AD? of GAL4 or LexA sad a catchingJpreying protein or peptide. It also has a nucleus ~.ocation signal. The catching-fnsiot~ protein is cvnseqneatly also transported into the yeast nucleus. If the catching protein and the baiting protein exposed at the BAS physically interact xith One another, then there is an increase in the statistical probability that the activating domain is located in the vicinity of the reporter gene promoter. This leads to an increase ~.n the transcription of the reporter genes, Whose extent is proportional to the strength of the interaction between the baiting and catching protein. The "Yeast Two-Hybrid System" can be used both for the quantitative analysis of knoaa bait/catch pairs and for the identification of unknown catching proteins or peptides_ Possible catthing proteins are e.g. a cDNA library or a combinatory peptide librazy.
Despite the above-described multiplicity of uses the "Yeast Two-Hybrid System"
suffers from limitations due to the transcription-dependent detection system.
They occur e.g. if the catching aad/nr baiting protein contains localization signals. These interfering localization signals are e.g. hydrophobic trans-membrane domains, such as occur in many membrane proteins. They lead to a transportation of the fusion protein into the cellular membrane, whilst the nucleus localization signal of the fusion protein is ignored. Therefore interactions with proteins having such transmembrane domains cannot be detec-ted. A further problem arises on screening for the analysis of cDNA libra-ries yr combinatory peptide libraries, which can only be carried out to a limited Qxtent. Due to their complexity (e. g. more than IO bilZ~,vn pos5xble variants far a decapQptide) the latter make efficient screening ittpvssible.
The analysis of possiblQ interaction partners has therefore hitherto tdken place in that a maximum of 50,000 transformed yeast cells per agar plate are plated out and incubated initially for 3 to 7 days in the incubator. This weans that a typical test of approximately 5 ~llion possible fusion protein pairs requires the use of 100 agar plates, which must be thoroughly tested.
Thus, the "Yeast Two-Hybrid System" involves high material, time, laboux and culture space costs for this screening process type. Iu addition, there x,s no known automated analysis process, which could reduce and standardize the high laboratory staff costs.
Thus, the problew of the present invention is to pxovide a method and a corresponding kit for the idQntification of interactions between proteins or peptides, which at least partly avoid the aforQmentfoned disadvantages and which can 3.n particular be carried out in an automated manner.
?iLTROD ARD ICIT F08 IDENTIFYING 119TERAGTIONS BE1'ilEEl~ PROTEINS OR PEPTIDES
The present invention relates to a method and to a kit for the identification or determination of interactions between prot~ias or peptides by means of fluorescence-resonance energy transfer.
The identificatiott_and analysis of interactions bets~reen different proteins or peptides or fragments thereof represents as important problem of biopiedical research and biotechnology. At the end of the 1980's a system W88 coo- .
sequently developed, which acquired great research signif~.cance under the name "Yeast Two-Nyhrid-System" (Fields et ai.,.Natnre, vol.. 340, pp z45~247, 1489). Ti~.ts system is based on the discovery that cellular transcription activators,. such as e.g. GAL4 or lexA from yeast could be deCOmposed in Lw0 f independent fvmtctloa domains. Both domains are normally a component .af a protein in the nucleus of the yeast cell, which binds to.specific actipatiag sequences~~of diffazeat target genes and regulates the transcription thereof.
Ons domain,~the~.DNA~~binding domain (8D), specifically binds to.a particular DHA target seqaeace (upstream activating seqaeace) in the vicinity of the target gene promoter. The oilier domain, the activatins domaf.u (AD), increases the transcription rate of the target gene by interaction pith the transcription initiation complex, which is bound to the protater of the target gene. In the "Yeast Two-Hpbrid System" this structure is utilized by transcription factors in modified form. The DNA binding domain (BD) of GAL4 or lesA is there expressed as a fuaioa protein with a "baiting protein. or peptide" in yeast cells. This fusion protein also has a nucleus location shgnai through which it is transported into the yeast nucleus. The.baiting-fusi.on protein is bound there to a target sequence (UAS), which in the yeast strain used is located in the viciaf.ty of prvmaters of two reporter genes (e. g. anxotrophic i~aarker (HIS3) and enzy~tatic mar$er (lacZ)). This leads to s'conStellatioa in which the baiting protein or peptide is exposed ~in direct. spatial proximity to the reporter gene promoter. Additionally a second fusion protein is expressed in the same yeast.cell. Zt Comprises the activating domain (AD? of GAL4 or LexA sad a catchingJpreying protein or peptide. It also has a nucleus ~.ocation signal. The catching-fnsiot~ protein is cvnseqneatly also transported into the yeast nucleus. If the catching protein and the baiting protein exposed at the BAS physically interact xith One another, then there is an increase in the statistical probability that the activating domain is located in the vicinity of the reporter gene promoter. This leads to an increase ~.n the transcription of the reporter genes, Whose extent is proportional to the strength of the interaction between the baiting and catching protein. The "Yeast Two-Hybrid System" can be used both for the quantitative analysis of knoaa bait/catch pairs and for the identification of unknown catching proteins or peptides_ Possible catthing proteins are e.g. a cDNA library or a combinatory peptide librazy.
Despite the above-described multiplicity of uses the "Yeast Two-Hybrid System"
suffers from limitations due to the transcription-dependent detection system.
They occur e.g. if the catching aad/nr baiting protein contains localization signals. These interfering localization signals are e.g. hydrophobic trans-membrane domains, such as occur in many membrane proteins. They lead to a transportation of the fusion protein into the cellular membrane, whilst the nucleus localization signal of the fusion protein is ignored. Therefore interactions with proteins having such transmembrane domains cannot be detec-ted. A further problem arises on screening for the analysis of cDNA libra-ries yr combinatory peptide libraries, which can only be carried out to a limited Qxtent. Due to their complexity (e. g. more than IO bilZ~,vn pos5xble variants far a decapQptide) the latter make efficient screening ittpvssible.
The analysis of possiblQ interaction partners has therefore hitherto tdken place in that a maximum of 50,000 transformed yeast cells per agar plate are plated out and incubated initially for 3 to 7 days in the incubator. This weans that a typical test of approximately 5 ~llion possible fusion protein pairs requires the use of 100 agar plates, which must be thoroughly tested.
Thus, the "Yeast Two-Hybrid System" involves high material, time, laboux and culture space costs for this screening process type. Iu addition, there x,s no known automated analysis process, which could reduce and standardize the high laboratory staff costs.
Thus, the problew of the present invention is to pxovide a method and a corresponding kit for the idQntification of interactions between proteins or peptides, which at least partly avoid the aforQmentfoned disadvantages and which can 3.n particular be carried out in an automated manner.
They lead to a transportation of the fusion pxotein into the cellular mem-brane, whilst the nucleus localization signal of the fusion proteia is ignored. Therefore interactions with proteins havi~tg such transmembrane domains cannot be detected. A further problem arises on screening far the analysis of eDPiA libraries or combinatory peptide libraries, which can only be carried out to a Limited extent. Due to their complexity (e. g. more than billioa possible variants for a decapeptide) the lattex make efficient screening iuipvssible. The analysis of possible interaction partners has therefore hitherto taken place in that a maximum of 50,000 transformed yeast cells per agar plate are plated out and incubated initially for 3 to 7 days in the incubator. This means that a typical test in the order of 5 million possible fusion protein pairs requires the use of 100 agar plates, which roust be thorpughly tested. Thus, the "Yeast Two-gyb~d System" involves high material, time, labour and culture space costs for the screening processes.
In addition, there is no known automated analysis process, which would reduce and standardise the high laboratory staff costs.
International patent application W0 97/27212 describes a screening method with which pegtide sequences are identified which measurably modify the phenotype of a cell. The method is based oa the measuretuent of the phenotype change after expression of the peptide in the cell. Initially no inter-actions of peptides or proteins are investigated. Only in a second step is it possible tv carry out a closer investigation of the identified, active peptide sequences. For thin purpose various Ialown methods can be alter-natively used, e_g. the absence of aormal cellular functions can be measured, a two-hybxid system in mammalian cells can be used or a fluorescence-resonance energy transfQr system can be e~uployrd.
The literature already describe souse test systems for investigating l~oWn interaction partners, which utilize fluorescence-resonance eaergy transfer.
For example, in international patent app~Cation WO 9I/12530 a fluorescence test is described for investigating interactions of specific binding partners, particularly With respect to immunoassays. European patent application EP
0242527 describes an aaalysis process for interacting analytes using the energy transfer system.
Amended sheet - 3a -Thus, the probie~ of the present invention is to provide a method and a corresponding kit for the identification of interactions between proteins or peptides, which at Least partly avoid the aforementioned disadvantages and whf.ch can in particular be carxied out i.n an automated manner_ This problem is solved by the subject matter of the claims and their wording is hereby made by reference into part of the content of the present descrip-tion_ Amended sheet This problem is in particular soloed by a method in which at least two proteins or peptides are coupled to different fluorescent components, the absorption and emission spectra of the fluorescent components overlapping in such a way that fluorescence-resonance energy tra~zsfer (FRET) is possible and the components are so brought together by an interaction between the proteins or peptides that FRET occurs and is measured. The genetic information far at least two fusion peptides or proteins having a peptide or pxotein fraction attd in each case one different fluorescent protein yr peptide is present in an expression systeta introduced into a host ceh, the emis8ion arid absorption Spectra of~the fluorescent preteins or peptides overlapping i~1 such a way that fluoresenCe-resonance energy transfer (FRET) occurs and is measured in the host cell.
Within the scope of the present invention the terms "protein" and "peptide"
are mutually interchangeable and contain an amino acid line-up of any length and complexity, i.e. dipeptides, vligopeptides, polypegtides, Complete pro-teins, ~ragments thereof, antibodies, domains, epitopes, etc. They can in particular be combinatory peptide libraries or the expression products of cDNA libraries. The protcins/peptides to be investigated for interactions are subject to no restrictions and can be freely chosen.
According to the invention, the term fluorescent component is in particular understood to mean a material or particles having a fluorescent marking at the surface. They can he latex particles or matrices, as acre of a conven-tional nature for automatic protein synthesis.
According to the invention coupling means a more or Less firm binding of the Amended sheet fluorescent component to the proteins) or peptide(s). This bond tends to be of a more adsorptive nature or can also be a covalent bond. in a pre-ferred embodiment the protein or peptide and the fluorescent component can be present in the form of a fusion protein or peptide. Coupli~xg can also take place by means of a linker. The latter covers connections of ah types suitable for linking two mplecules_ The method according to the invention is inter alxa based on the known phenomenon of fluorescence-resonance energy transfer (FRET), diagrammatically represented i.n fig. 1. A fluorescent molecule absorbs photons with a charxsc-teristic wavelength and liberates again the thus absorbed energy within a very short time by the emission of photons, which gives rise to a measurable fluorescence. Since during this process there is a certain energy loss by heat evolution, the e~.tted photon has a characteristic, reduced energy content and therefore a modified wavelength cotapared with the previously absorbed photon (Stokes shl.ft). Both parameters, namely the absorption wavelength and the Stokes shift (and the emission wavelength dependent there-on) are charatteri-stic parameters of any fluorescent molecule and are a func-tion of the characteristics thereof. A fluorescence-resonance energy trans-fer tan be measured if two fluorescent molecules interact in a mixture with one another and the absorption wave~,ength of one overlaps with the emission wavelength of the other. In this connection Amended sheet enzymatic ligation and cloned in an expression vector using standard methods.
It ix also possible to use here a randoptly complex population o~ baiting and catching proteins in order to obtain a library of possible interaction partners. According to the invention genetic information and coding DNA
sequence means cDNA or genomic DNA, preferably cDNA.
Known, conventional vectors can be used as expression vectors. However, they should permit a separate selection of "baiting protein vectors" and "catching protein vectors" in the ~vst cells, e.g. by different antibiotic resistance genes (e. g. ampicillin, kanamycin, chloroamphenicol, streptomycin, tetra-cyclines, sulphonamides) oz different auxotrophic markers (e.g. LEU2, 8IS3 or TRPl)_ However, it is also possible to combine both fusion proteins together in a bicistronic expression vector. 5nitable expression vectors for the expression in E.coli are e.g. pGEMEIC, pUC derivatives, pGEg-2T, pET3b, pQE8 or pQE42, for the expression in yeast pY100, Ycpadl, pGBT9 or pGAD424, for the expression in animal cells pKCR, pEFBOS, cDMB and pCEV4. In parti.CUlar, the Baculovirus expression vector pAcSGNt-A is suitablQ for expression in insect ce118.
The expert is also aware of methods and host cells for expressing the expression vector and the fusion protein coded by it. Examples of such cells are the E.cali strains HB10I, DH1, x1776, JM1O1, Jm109, B121 and SG13009, the yeast strain Saccharou~Ces cerevisiae, Schi2osaccharo~ces pombe, Y 190, CG1945, EGY48 or HF7, the animal cells L, 3T3, FM3A, CHO, COs, Vero and Hela and the insect cells Sf9. Suitable cells, preferably yeast cells, are (sequentially or synchronously) transformed with the expression vector yr vectors and preferably propagated under double selection.
The fluorescence of the cells is then excited at the absorption maximum of one of the two fluorescent proteins (preferably at the maximum of FP-A), measured at the emission maximum of the other fluorescent protein (preferably of FP-B) and the ~ueasnred result is used as a selectf.on criterion in the ipso, lation and cloning of cells contafning a potential interaction pair. This measurement i.s calibrated bQforehand by cells in which PP-A and FP-B are expressed cloned in different vectors, but witheut fusing with a further protein domain (negative control). The positive control can be constituted by a fusion protein in which FP-A and FP-B (~rithont baiting or catching pro-tein) are coupled to one another in the above-described manner and con-sequently there is a maxiirium spatial proximity between FP-A and FP-B, Fluorescence is preferably exca.ted by laser. FRET measurement takes place using suitable filter combinations, which ran be chosen by the expert as a function of the absorption and emission maxima of the fluorescent proteins used_ In accordance with the bases of FRET each interaction between the fusion proteins mast lead to a FRET rise, which can be detected after appropriate calibration of the system. For the investigation of known bait/
catch pairs this detection preferably takes place by fluorescence microscopy.
For the screening of cDNA libraries ox combinatory peptide libraries measure-ment preferably takes place by fluorescence-controlled cell sorting (FACS).
For further identification of the interaction partners determined by FRET
they are plated out on agar plates and plasmide-DtiA is iso).ated from the individual colonies and can then be sequenced using standard methods.
The method according to the invention has e.g. the following preferred applications:
- A known pair of possible interaction partners is jointly cxgressed in a host cell and the resulting fluorescence spectrum is compared with the nega-tive and positive controls or other interaction partners.
- A baiting-fusion protein is transformed or tx~ansfected with a randomly complex mixture of catching-fusion proteins (jointly or sequentially) in suitable host cells and preferably in such a way that each cell only carries one defined pair of possible interaction partners or a small group of such pairs. From the resulting host cell populations are then isolated the Cells which have a significant FRET and consequently e~cpress a potential inter-action pair_ By isolating the eacprassion vectors Contained xn these cells it is easily possible to determine the sequence of the participating interacta.on partners. The mixture of catching proteins could e_g. be made o~ cpNA
libraries or combinatory peptide libraries.
g _ The baiting protein could also be represented by a population of different proteins_ It is conseQuently e.g. possible to determine an interaction matrf.x, e.g. in that a cbNA library is searched for possible interaction pairs between unknown proteins.
- A known pair of interacting fusion proteins is jointly expressed in host cells and the fluorescence transfer between the two fusion proteins is deter-mined_ Then a third protein or peptide is expressed in the same host cells and its influence on FRET is trade an analysis criterion. This makes it pos3-ible to datvrmine proteins or peptides which disturb or intensify an existing interaction between known interaction partners.
The advantages of the method according to the invQntion axe that unlike is the "Yeast Two-Hybrid System" it is independent of transcr~,ption-regulating mechanisms and the location of fusion proteins in the nucleus is nn~.mportant.
Thus, e.g. also intact membrane proteins can be used as baiting proteins.
The possibility of an automated cell, sorting additionally exists, which permits the obtaW ing of a high screening complexity. Thxough the combin-ation of a pharmacologically interesting target sequence and a combinatory peptide library it is possible to screen several million peptide ligands per time unit. Compared with known methods with which thin task might not be performable, the method according to the invention is characterized by an enormous time and labon.r saving.
The kit according to the invention (test IQt) is described in claim 8.
Preferred embodiments of this kit can be gathered from the dependent clairus 9 to 16.. The wording of claims 8 to 16 xs hereby made by reference into part of the content of this description. Express reference is made to previous parts of the description directly or indirectly linked with the features of claims 8 to 16_ The claimed kit is used fOx performing the claimed method in a particularly simple manner. the kit preferably contains in a suitable container a first vector, which carries the host cell-optimized coding sequence of a FRET donor (under the control of a suitable promoter) and in which there is a cloning _ c~
possibility fer the introduction of a coding sequence, which codes for a first test protein/test peptide (i.e. a baiting or catching protein or peptide).
There is also an associated second vector with host cell-optimized coding sequence for the associated FRET acceptor and cloning possibility for the coding sequence of a second test protein/test peptide (corresponding inter-action partner for the bait-catch pair zo be formed).
The coding sequences for the test proteins/test peptides in the first and/or second vQCtor may already be present. Preferably also Integrated CDNA
libraries or combinatory oligonncleotide Libraries are contained. In such cases e.g. a cbftA library (e.g. from the human brain) is present.with one of the FRET partners fused in a first vector and a second vector in which the user can clone an owa protei.n/peptide, e.g. as bait, with the other FRET
partner, The invention is described hereinafter relat~.ve to the drawings, wherein shoo:
Fig. 1 A diagrammatic representation of fluorescence~resonance energy transfer (FILET) .
Fig. 2 A diagrammatic representation of the energy shift.
Fig. 3 A diagran~uatic representation of FRET by mesas of the interact~.ou of baiting protein with catching protein.
Fig, 4. A gene map Of the expression vector pGBT9.
Fig. 5 The gene map of the e~cpression vector pGAD42~.
The inventf.on is described by means of the followiag examples.
E~CAMpLE I
Initially a suitable vector system is formed. For this purpose and for reasons of siuiplicity use is made of the plasmides pG8T9 (~K I605-A), pGAD424 (#'K1605-B) of Clontech (Palo Alto, US) and the plasmides pRSET
$-P4-3 and pRSET $-S65T (Helm and Tsien, Curz-ent Biology 1996, 6: 178-182)_ pGBT9 is a possible "baiting protein vector" of the Yeast Two-Hybrid System marketed by Clontech. The vector contains an ampicilLin resistance gene for selection in bacteria, a TRP1 gene for anxotrophic selection in yeast and an expression cassette for the DNA-binding domain of the GAL4 transcription factor (fig. 4, Clontech Hatchu~aker "GAIk Two-gybrid Vectors $andbook, ~pT3062-1) wader the control of an alcohol dehyd=ogenase promoter (ADH1) for expression in yeast. The GAL4-DNA-binding domain is an essential, com-ponent of the Yeast Two-Hybrid System.
For the purpose of the invention the GAL4 DNA-binding domain together with its nucleus location signal is removed, is that it is cut out by moans of a HinDIII/EcoRI restriction digestion.
pGAD4~24 is a possible "catching protein vector" of the Yeast Two-Hybrid System (fig. 5). This vector has a similar structure to pGBT9, except that instead of the TBPI ~uarker it contains a L$U2 gene for selection in yeast and in place of the GAL4 DNA-binding domain ~t contains the GAL4 activating domain.
For the purpose of the invention also from this vector is removed by a HiaDIYI/EcoRI digestion the GAL4 activating doma~.n. For this purpose initially further HinDIII restriction sites present in the vector are removed by partial digestion, filling the restricting site with Pfu polymerase and subsequent "blunt end Iigation".
Suitable coding sequences of GFP variants S65T and P4-3 (Heim and Tsien, Current Biology 6: 178-182, 1996) are now amplified by PCR and provided at their S' and 3' ends With AinDIxI or EcoRI restriction sites. For this purpose use is made of the oli.gonncleotide primers GFP-Eco RI-3'(3'-CGGGAATTCTTTGTATAGTTCATCCAT-3'), and GFP-HinDIII-S'-TCCAAGCTTATGAGTAAAGGAGAAGAACTT-3') (Carl Both GmbR, Karlsrnhe). As the coding sequences of P4-3 and S65T are identical in their 5' and 3'-terminal e.g, in that a cDNA library is searched for possible interaction pairs between unknown proteins.
- A known pair of interacting fusion proteins is jointly expressed in host cells and the fluorescence transfer between the two fusion proteins is detex.-wined. Then a third protein or peptide is expressed in the same host cells and its influence on FRET is made an analysis criterion. This makes it poss-ible to determine proteins or peptides which disturb or intensify an existing interaction between known interaction partners.
The advantages of the method according to the invention are that unlike in the "Yeast Two-hybrid System" it is independent of transcription-regulating mechanisms and the location of fusion proteins in the nucleus i.s unimportant.
Thus, e.g. also intact membrane proteins can be used as baiting proteins.
The possibility of an automated cell sorting additionally e~.sts, which permits the obtaining of a high screening complexity. Through the combin-ation of a pharmacologically interesting target sequence and a combinatory peptide library it is possible to screen several million peptide ~.igands per t3~e LZnit. Compared with known methods with which this task might not be performable, the method according to the invention is characterized by an enormous time and labour saving.
The kit according to the itwention (test kit) is described in claim 7.
Preferred embodiments of this kit can be gathered from the dependent claims 8 to I5. The wording of claims 7 to I5 is hereby taade by reference into part of the content of this description. Express reference is trade to previous parts of the description directly or indirectly linked with the features of claims 7 to 15.
The claimed kit is used for performing the claimed method in a particularly simple manner. The kd.t preferably contains iu a suitable container a first vector, which carrzes the host cell-optimized coding sequence of a FRET
donor (under the control of a suitable prouaoter) and in which there is a cloning possibility for the introduction of a coding sequence, which codes Amended sheet -lz-for a first test protein/test peptide (i.e_ a baiting or catching protein or peptide).
There is also an associated second vector with host cell-optimized coding sequence for the associated FRET acceptor and cloning possibility for the coding sequence of a second test protein/test peptide (corresponding inter-action partner for the bait-catch pair to be formed).
The coding sequences for the test proteias/test peptides in the first and/ox second vector map already be present. Preferably also 3.ntegrated cDIZA
libraries or combinatory oligonucleotide 7.ibraries are contained. In such cases e.g. a cDNA library (e.g. from the human brain) is present with one of the FRET partners fused in a first vector and a second vector in which the user can clone an own protein/peptide, c.g. as bait, with the other FRET
partner.
The invention is described hereinafter relative to the dxawings, wherein show:
Amended sheet cells, once with the baiting plasmide pGBT-BFP-P2g.2 and a further time with the empty baiting plasmide vector pGBT-BFP. The fluorescence spectra of the tr~m transformants are then compared is order to exclude those interaction candidates rahich interact directly with the blue fluorescent protein, but not with the baiting protein.
Candidates checked in this way are they plated out again on selective minimum mediuta plates and the sequence of the potentially interacting catching domain is determined by standard molecular biology methods.
EgAMpLE 2 Use is made of two vectors, the first being selected from the commercially ~evailable expression vectors fvr EBfP or EGFP, e.g. pEBFP-N1, pE$FP-NZ, pEBFP-N3, pEBFP-CI, pEBFP-CZ, pEBFP-C3, pECFP, pECFP-GI (Clontech) and the SeCOnd from the commercially available expression vectoxs for EGFP or EYFP, e.g. pEGFP-NI, pEGFP-N2, pEGFP-N3, pEGFp-C1, pEGFp-C2, pEGFp-C3, pEYFP-N1, pEYFP N2, pEYFP-N3, pEYFP-C1, pEYFP-CZ, pEYfP-G3.
In one of the tyro vectors a DNA, sequence is cloned in the usual way and this codes a baiting protein, so that the baiting protein is read off as a fusion protein wa.th the fluorescent protein. In the other vector a cDNA library ox a combinatory oligonucleotide library is Cloned, so that the second fluore-scent protein is read off as a fusion protein with in each case a protein fragment or combinatory peptide. Both vectors together are then transfected in known manner iu suitable mammalian cells, e.g. COS-7, NG-108, H1H/3T3, etc.
Following an adequate incubation time the cehs are then analyzed by FREx microscopy (see Clegg, in "Fluorescence Imaging Spectroscopy and tsicroscopy", pp I79-236, John Wiley & Sons, 1996) or in the above-described manner are individualized by flnorescence,activated cell sorting (FRCS). The cells in which FRET occurs are then used in the conventional manner, e.g. by cytoplast iso7.ati.on with a miervpipette conventionally used in electrophps5.ology and subseqaQnt RT-PCR used fvr determining the basic catch sequences.
In addition, there is no known automated analysis process, which would reduce and standardise the high laboratory staff costs.
International patent application W0 97/27212 describes a screening method with which pegtide sequences are identified which measurably modify the phenotype of a cell. The method is based oa the measuretuent of the phenotype change after expression of the peptide in the cell. Initially no inter-actions of peptides or proteins are investigated. Only in a second step is it possible tv carry out a closer investigation of the identified, active peptide sequences. For thin purpose various Ialown methods can be alter-natively used, e_g. the absence of aormal cellular functions can be measured, a two-hybxid system in mammalian cells can be used or a fluorescence-resonance energy transfQr system can be e~uployrd.
The literature already describe souse test systems for investigating l~oWn interaction partners, which utilize fluorescence-resonance eaergy transfer.
For example, in international patent app~Cation WO 9I/12530 a fluorescence test is described for investigating interactions of specific binding partners, particularly With respect to immunoassays. European patent application EP
0242527 describes an aaalysis process for interacting analytes using the energy transfer system.
Amended sheet - 3a -Thus, the probie~ of the present invention is to provide a method and a corresponding kit for the identification of interactions between proteins or peptides, which at Least partly avoid the aforementioned disadvantages and whf.ch can in particular be carxied out i.n an automated manner_ This problem is solved by the subject matter of the claims and their wording is hereby made by reference into part of the content of the present descrip-tion_ Amended sheet This problem is in particular soloed by a method in which at least two proteins or peptides are coupled to different fluorescent components, the absorption and emission spectra of the fluorescent components overlapping in such a way that fluorescence-resonance energy tra~zsfer (FRET) is possible and the components are so brought together by an interaction between the proteins or peptides that FRET occurs and is measured. The genetic information far at least two fusion peptides or proteins having a peptide or pxotein fraction attd in each case one different fluorescent protein yr peptide is present in an expression systeta introduced into a host ceh, the emis8ion arid absorption Spectra of~the fluorescent preteins or peptides overlapping i~1 such a way that fluoresenCe-resonance energy transfer (FRET) occurs and is measured in the host cell.
Within the scope of the present invention the terms "protein" and "peptide"
are mutually interchangeable and contain an amino acid line-up of any length and complexity, i.e. dipeptides, vligopeptides, polypegtides, Complete pro-teins, ~ragments thereof, antibodies, domains, epitopes, etc. They can in particular be combinatory peptide libraries or the expression products of cDNA libraries. The protcins/peptides to be investigated for interactions are subject to no restrictions and can be freely chosen.
According to the invention, the term fluorescent component is in particular understood to mean a material or particles having a fluorescent marking at the surface. They can he latex particles or matrices, as acre of a conven-tional nature for automatic protein synthesis.
According to the invention coupling means a more or Less firm binding of the Amended sheet fluorescent component to the proteins) or peptide(s). This bond tends to be of a more adsorptive nature or can also be a covalent bond. in a pre-ferred embodiment the protein or peptide and the fluorescent component can be present in the form of a fusion protein or peptide. Coupli~xg can also take place by means of a linker. The latter covers connections of ah types suitable for linking two mplecules_ The method according to the invention is inter alxa based on the known phenomenon of fluorescence-resonance energy transfer (FRET), diagrammatically represented i.n fig. 1. A fluorescent molecule absorbs photons with a charxsc-teristic wavelength and liberates again the thus absorbed energy within a very short time by the emission of photons, which gives rise to a measurable fluorescence. Since during this process there is a certain energy loss by heat evolution, the e~.tted photon has a characteristic, reduced energy content and therefore a modified wavelength cotapared with the previously absorbed photon (Stokes shl.ft). Both parameters, namely the absorption wavelength and the Stokes shift (and the emission wavelength dependent there-on) are charatteri-stic parameters of any fluorescent molecule and are a func-tion of the characteristics thereof. A fluorescence-resonance energy trans-fer tan be measured if two fluorescent molecules interact in a mixture with one another and the absorption wave~,ength of one overlaps with the emission wavelength of the other. In this connection Amended sheet enzymatic ligation and cloned in an expression vector using standard methods.
It ix also possible to use here a randoptly complex population o~ baiting and catching proteins in order to obtain a library of possible interaction partners. According to the invention genetic information and coding DNA
sequence means cDNA or genomic DNA, preferably cDNA.
Known, conventional vectors can be used as expression vectors. However, they should permit a separate selection of "baiting protein vectors" and "catching protein vectors" in the ~vst cells, e.g. by different antibiotic resistance genes (e. g. ampicillin, kanamycin, chloroamphenicol, streptomycin, tetra-cyclines, sulphonamides) oz different auxotrophic markers (e.g. LEU2, 8IS3 or TRPl)_ However, it is also possible to combine both fusion proteins together in a bicistronic expression vector. 5nitable expression vectors for the expression in E.coli are e.g. pGEMEIC, pUC derivatives, pGEg-2T, pET3b, pQE8 or pQE42, for the expression in yeast pY100, Ycpadl, pGBT9 or pGAD424, for the expression in animal cells pKCR, pEFBOS, cDMB and pCEV4. In parti.CUlar, the Baculovirus expression vector pAcSGNt-A is suitablQ for expression in insect ce118.
The expert is also aware of methods and host cells for expressing the expression vector and the fusion protein coded by it. Examples of such cells are the E.cali strains HB10I, DH1, x1776, JM1O1, Jm109, B121 and SG13009, the yeast strain Saccharou~Ces cerevisiae, Schi2osaccharo~ces pombe, Y 190, CG1945, EGY48 or HF7, the animal cells L, 3T3, FM3A, CHO, COs, Vero and Hela and the insect cells Sf9. Suitable cells, preferably yeast cells, are (sequentially or synchronously) transformed with the expression vector yr vectors and preferably propagated under double selection.
The fluorescence of the cells is then excited at the absorption maximum of one of the two fluorescent proteins (preferably at the maximum of FP-A), measured at the emission maximum of the other fluorescent protein (preferably of FP-B) and the ~ueasnred result is used as a selectf.on criterion in the ipso, lation and cloning of cells contafning a potential interaction pair. This measurement i.s calibrated bQforehand by cells in which PP-A and FP-B are expressed cloned in different vectors, but witheut fusing with a further protein domain (negative control). The positive control can be constituted by a fusion protein in which FP-A and FP-B (~rithont baiting or catching pro-tein) are coupled to one another in the above-described manner and con-sequently there is a maxiirium spatial proximity between FP-A and FP-B, Fluorescence is preferably exca.ted by laser. FRET measurement takes place using suitable filter combinations, which ran be chosen by the expert as a function of the absorption and emission maxima of the fluorescent proteins used_ In accordance with the bases of FRET each interaction between the fusion proteins mast lead to a FRET rise, which can be detected after appropriate calibration of the system. For the investigation of known bait/
catch pairs this detection preferably takes place by fluorescence microscopy.
For the screening of cDNA libraries ox combinatory peptide libraries measure-ment preferably takes place by fluorescence-controlled cell sorting (FACS).
For further identification of the interaction partners determined by FRET
they are plated out on agar plates and plasmide-DtiA is iso).ated from the individual colonies and can then be sequenced using standard methods.
The method according to the invention has e.g. the following preferred applications:
- A known pair of possible interaction partners is jointly cxgressed in a host cell and the resulting fluorescence spectrum is compared with the nega-tive and positive controls or other interaction partners.
- A baiting-fusion protein is transformed or tx~ansfected with a randomly complex mixture of catching-fusion proteins (jointly or sequentially) in suitable host cells and preferably in such a way that each cell only carries one defined pair of possible interaction partners or a small group of such pairs. From the resulting host cell populations are then isolated the Cells which have a significant FRET and consequently e~cpress a potential inter-action pair_ By isolating the eacprassion vectors Contained xn these cells it is easily possible to determine the sequence of the participating interacta.on partners. The mixture of catching proteins could e_g. be made o~ cpNA
libraries or combinatory peptide libraries.
g _ The baiting protein could also be represented by a population of different proteins_ It is conseQuently e.g. possible to determine an interaction matrf.x, e.g. in that a cbNA library is searched for possible interaction pairs between unknown proteins.
- A known pair of interacting fusion proteins is jointly expressed in host cells and the fluorescence transfer between the two fusion proteins is deter-mined_ Then a third protein or peptide is expressed in the same host cells and its influence on FRET is trade an analysis criterion. This makes it pos3-ible to datvrmine proteins or peptides which disturb or intensify an existing interaction between known interaction partners.
The advantages of the method according to the invQntion axe that unlike is the "Yeast Two-Hybrid System" it is independent of transcr~,ption-regulating mechanisms and the location of fusion proteins in the nucleus is nn~.mportant.
Thus, e.g. also intact membrane proteins can be used as baiting proteins.
The possibility of an automated cell, sorting additionally exists, which permits the obtaW ing of a high screening complexity. Thxough the combin-ation of a pharmacologically interesting target sequence and a combinatory peptide library it is possible to screen several million peptide ligands per time unit. Compared with known methods with which thin task might not be performable, the method according to the invention is characterized by an enormous time and labon.r saving.
The kit according to the invention (test IQt) is described in claim 8.
Preferred embodiments of this kit can be gathered from the dependent clairus 9 to 16.. The wording of claims 8 to 16 xs hereby made by reference into part of the content of this description. Express reference is made to previous parts of the description directly or indirectly linked with the features of claims 8 to 16_ The claimed kit is used fOx performing the claimed method in a particularly simple manner. the kit preferably contains in a suitable container a first vector, which carries the host cell-optimized coding sequence of a FRET donor (under the control of a suitable promoter) and in which there is a cloning _ c~
possibility fer the introduction of a coding sequence, which codes for a first test protein/test peptide (i.e. a baiting or catching protein or peptide).
There is also an associated second vector with host cell-optimized coding sequence for the associated FRET acceptor and cloning possibility for the coding sequence of a second test protein/test peptide (corresponding inter-action partner for the bait-catch pair zo be formed).
The coding sequences for the test proteins/test peptides in the first and/or second vQCtor may already be present. Preferably also Integrated CDNA
libraries or combinatory oligonncleotide Libraries are contained. In such cases e.g. a cbftA library (e.g. from the human brain) is present.with one of the FRET partners fused in a first vector and a second vector in which the user can clone an owa protei.n/peptide, e.g. as bait, with the other FRET
partner, The invention is described hereinafter relat~.ve to the drawings, wherein shoo:
Fig. 1 A diagrammatic representation of fluorescence~resonance energy transfer (FILET) .
Fig. 2 A diagrammatic representation of the energy shift.
Fig. 3 A diagran~uatic representation of FRET by mesas of the interact~.ou of baiting protein with catching protein.
Fig, 4. A gene map Of the expression vector pGBT9.
Fig. 5 The gene map of the e~cpression vector pGAD42~.
The inventf.on is described by means of the followiag examples.
E~CAMpLE I
Initially a suitable vector system is formed. For this purpose and for reasons of siuiplicity use is made of the plasmides pG8T9 (~K I605-A), pGAD424 (#'K1605-B) of Clontech (Palo Alto, US) and the plasmides pRSET
$-P4-3 and pRSET $-S65T (Helm and Tsien, Curz-ent Biology 1996, 6: 178-182)_ pGBT9 is a possible "baiting protein vector" of the Yeast Two-Hybrid System marketed by Clontech. The vector contains an ampicilLin resistance gene for selection in bacteria, a TRP1 gene for anxotrophic selection in yeast and an expression cassette for the DNA-binding domain of the GAL4 transcription factor (fig. 4, Clontech Hatchu~aker "GAIk Two-gybrid Vectors $andbook, ~pT3062-1) wader the control of an alcohol dehyd=ogenase promoter (ADH1) for expression in yeast. The GAL4-DNA-binding domain is an essential, com-ponent of the Yeast Two-Hybrid System.
For the purpose of the invention the GAL4 DNA-binding domain together with its nucleus location signal is removed, is that it is cut out by moans of a HinDIII/EcoRI restriction digestion.
pGAD4~24 is a possible "catching protein vector" of the Yeast Two-Hybrid System (fig. 5). This vector has a similar structure to pGBT9, except that instead of the TBPI ~uarker it contains a L$U2 gene for selection in yeast and in place of the GAL4 DNA-binding domain ~t contains the GAL4 activating domain.
For the purpose of the invention also from this vector is removed by a HiaDIYI/EcoRI digestion the GAL4 activating doma~.n. For this purpose initially further HinDIII restriction sites present in the vector are removed by partial digestion, filling the restricting site with Pfu polymerase and subsequent "blunt end Iigation".
Suitable coding sequences of GFP variants S65T and P4-3 (Heim and Tsien, Current Biology 6: 178-182, 1996) are now amplified by PCR and provided at their S' and 3' ends With AinDIxI or EcoRI restriction sites. For this purpose use is made of the oli.gonncleotide primers GFP-Eco RI-3'(3'-CGGGAATTCTTTGTATAGTTCATCCAT-3'), and GFP-HinDIII-S'-TCCAAGCTTATGAGTAAAGGAGAAGAACTT-3') (Carl Both GmbR, Karlsrnhe). As the coding sequences of P4-3 and S65T are identical in their 5' and 3'-terminal e.g, in that a cDNA library is searched for possible interaction pairs between unknown proteins.
- A known pair of interacting fusion proteins is jointly expressed in host cells and the fluorescence transfer between the two fusion proteins is detex.-wined. Then a third protein or peptide is expressed in the same host cells and its influence on FRET is made an analysis criterion. This makes it poss-ible to determine proteins or peptides which disturb or intensify an existing interaction between known interaction partners.
The advantages of the method according to the invention are that unlike in the "Yeast Two-hybrid System" it is independent of transcription-regulating mechanisms and the location of fusion proteins in the nucleus i.s unimportant.
Thus, e.g. also intact membrane proteins can be used as baiting proteins.
The possibility of an automated cell sorting additionally e~.sts, which permits the obtaining of a high screening complexity. Through the combin-ation of a pharmacologically interesting target sequence and a combinatory peptide library it is possible to screen several million peptide ~.igands per t3~e LZnit. Compared with known methods with which this task might not be performable, the method according to the invention is characterized by an enormous time and labour saving.
The kit according to the itwention (test kit) is described in claim 7.
Preferred embodiments of this kit can be gathered from the dependent claims 8 to I5. The wording of claims 7 to I5 is hereby taade by reference into part of the content of this description. Express reference is trade to previous parts of the description directly or indirectly linked with the features of claims 7 to 15.
The claimed kit is used for performing the claimed method in a particularly simple manner. The kd.t preferably contains iu a suitable container a first vector, which carrzes the host cell-optimized coding sequence of a FRET
donor (under the control of a suitable prouaoter) and in which there is a cloning possibility for the introduction of a coding sequence, which codes Amended sheet -lz-for a first test protein/test peptide (i.e_ a baiting or catching protein or peptide).
There is also an associated second vector with host cell-optimized coding sequence for the associated FRET acceptor and cloning possibility for the coding sequence of a second test protein/test peptide (corresponding inter-action partner for the bait-catch pair to be formed).
The coding sequences for the test proteias/test peptides in the first and/ox second vector map already be present. Preferably also 3.ntegrated cDIZA
libraries or combinatory oligonucleotide 7.ibraries are contained. In such cases e.g. a cDNA library (e.g. from the human brain) is present with one of the FRET partners fused in a first vector and a second vector in which the user can clone an own protein/peptide, c.g. as bait, with the other FRET
partner.
The invention is described hereinafter relative to the dxawings, wherein show:
Amended sheet cells, once with the baiting plasmide pGBT-BFP-P2g.2 and a further time with the empty baiting plasmide vector pGBT-BFP. The fluorescence spectra of the tr~m transformants are then compared is order to exclude those interaction candidates rahich interact directly with the blue fluorescent protein, but not with the baiting protein.
Candidates checked in this way are they plated out again on selective minimum mediuta plates and the sequence of the potentially interacting catching domain is determined by standard molecular biology methods.
EgAMpLE 2 Use is made of two vectors, the first being selected from the commercially ~evailable expression vectors fvr EBfP or EGFP, e.g. pEBFP-N1, pE$FP-NZ, pEBFP-N3, pEBFP-CI, pEBFP-CZ, pEBFP-C3, pECFP, pECFP-GI (Clontech) and the SeCOnd from the commercially available expression vectoxs for EGFP or EYFP, e.g. pEGFP-NI, pEGFP-N2, pEGFP-N3, pEGFp-C1, pEGFp-C2, pEGFp-C3, pEYFP-N1, pEYFP N2, pEYFP-N3, pEYFP-C1, pEYFP-CZ, pEYfP-G3.
In one of the tyro vectors a DNA, sequence is cloned in the usual way and this codes a baiting protein, so that the baiting protein is read off as a fusion protein wa.th the fluorescent protein. In the other vector a cDNA library ox a combinatory oligonucleotide library is Cloned, so that the second fluore-scent protein is read off as a fusion protein with in each case a protein fragment or combinatory peptide. Both vectors together are then transfected in known manner iu suitable mammalian cells, e.g. COS-7, NG-108, H1H/3T3, etc.
Following an adequate incubation time the cehs are then analyzed by FREx microscopy (see Clegg, in "Fluorescence Imaging Spectroscopy and tsicroscopy", pp I79-236, John Wiley & Sons, 1996) or in the above-described manner are individualized by flnorescence,activated cell sorting (FRCS). The cells in which FRET occurs are then used in the conventional manner, e.g. by cytoplast iso7.ati.on with a miervpipette conventionally used in electrophps5.ology and subseqaQnt RT-PCR used fvr determining the basic catch sequences.
Claims (15)
1. Method for identifying peptide or protein interaction partners, in which at least two peptides or proteins are coupled to different fluorescent components, in which the absorption and emission spectra of the fluorescent components overlap in such a way that fluorescence-resonance energy transfer is possible and the fluorescence components can be brought together by an interaction between the proteins or peptides in such a way that FRET occurs and is measured, characterized in that peptides or proteins and fluorescent components in the form of fusion peptides or proteins are present and their genetic information is introduced into host cells in an expression system and FRET is measured there.
2. Method according to claim 1, wherein the fluorescent components with overlapping emission and absorption spectra are blue fluorescent protein and green fluorescent protein from Acquorea victoria.
3. Method according to claim 1 or 2, wherein the host cells are yeast cells.
4. Method according to one of the claims 1 to 3, wherein the measurement of the fluorescence-resonance energy transfer takes place by means of fluorescence microscopy or fluorescence-controlled cell sorting (FACS).
5. Method according to one of the claims 1 to 4, wherein fluorescence excitation takes place by laser.
6. Method according to one of the claims 1 to 5, wherein the protein or peptide fraction of the fusion protein or peptide comes from a combinatory peptide library or is the expression product of a cDNA library.
7. Kit for identifying interactions between proteins or peptides, comprising a) at least one first vector with - a coding sequence for a first fluorescent protein er peptide and - a cloning site by means of which it is possible to introduce a coding sequence for a first test protein or test peptide and b) at least one second vector with - a coding sequence for a second fluorescent protein or peptide, whose absorption spectrum overlaps with the emission spectrum of the first fluorescent protein or peptide in such a way that FRET occurs and - a cloning site by means of which one coding sequence for a second test protein or test peptide can be introduced.
8. Kit according to claim 7, characterized in that the coding sequences for the first and second fluorescent protein or peptide are sequences optimized for a specific host cell.
9. Kit according to claim 7 or 8, characterized in that the first vector and second vector comprise a promoter able to function in a specific host cell.
10. Kit according to one of the claims 7 to 9, characterized in that the first vector and/or the second vector has at least one marker gene, whose expression in the host cell permits a selection of the cells containing the particular vector.
11. Kit according to one of the claims 7 to 10, characterized in that the coding sequence for the first test protein/test peptide and/or the coding sequence for the second test protein/test peptide is already introduced at the particular cloning site.
12. Kit according to claim 11, characterized in that the coding sequence is a so-called nucleotide library, preferably a cDNA library.
13. Kit according to one of the claims 7 to 12, characterized in that the first vector and/or the second vector is a plasmide, the corresponding cloning site preferably being a restriction detection site.
14. Kit according to one of the claims 7 to 13, characterized in that it additionally comprises a quantity of a suitable host cell type, in which the first vector and/or the second vector can be expressed.
15. Kit according to claim 14, characterized in that the host cell is a yeast cell.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19737562.6 | 1997-08-28 | ||
| DE19737562A DE19737562A1 (en) | 1997-08-28 | 1997-08-28 | Method for the identification of interactions between proteins or peptides |
| PCT/EP1998/005458 WO1999012033A2 (en) | 1997-08-28 | 1998-08-28 | Method and kit for identifying interactions between proteins or peptides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2301633A1 true CA2301633A1 (en) | 1999-03-11 |
Family
ID=7840483
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002301633A Abandoned CA2301633A1 (en) | 1997-08-28 | 1998-08-28 | Method and kit for identifying interactions between proteins or peptides |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1007968B1 (en) |
| JP (1) | JP2001515208A (en) |
| AT (1) | ATE210828T1 (en) |
| AU (1) | AU9437598A (en) |
| CA (1) | CA2301633A1 (en) |
| DE (3) | DE19737562A1 (en) |
| WO (1) | WO1999012033A2 (en) |
Cited By (1)
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|---|---|---|---|---|
| WO2005050182A1 (en) * | 2003-11-19 | 2005-06-02 | Dimerix Biosciences Pty Ltd | Resonance energy transfer assay system for multi-component detection |
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| DE19829495A1 (en) * | 1998-07-02 | 2000-01-05 | Jacques Paysan | Reagents and their application for the investigation of interactions between cellular molecules and their localization in cells |
| EP1325148A1 (en) * | 2000-09-29 | 2003-07-09 | Universität Zürich | Method and kit for detecting membrane protein - protein interactions |
| US20080119423A1 (en) * | 2001-12-31 | 2008-05-22 | Shuster Samuel J | Methods and Materials For Modulating p2x2 |
| DE10234739A1 (en) * | 2002-07-30 | 2004-02-12 | Universitätsklinikum Hamburg-Eppendorf | New interfering splice variant of human p53 protein, useful for identifying modulators of p53 function, and potential anticancer agents |
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| US4868103A (en) * | 1986-02-19 | 1989-09-19 | Enzo Biochem, Inc. | Analyte detection by means of energy transfer |
| GB8916806D0 (en) * | 1989-07-22 | 1989-09-06 | Univ Wales Medicine | Modified proteins |
| US5342789A (en) * | 1989-12-14 | 1994-08-30 | Sensor Technologies, Inc. | Method and device for detecting and quantifying glucose in body fluids |
| GB9003593D0 (en) * | 1990-02-16 | 1990-04-11 | Pa Consulting Services | Improvements in or relating to fluorescence assays |
| JP3448090B2 (en) * | 1994-02-16 | 2003-09-16 | 浜松ホトニクス株式会社 | Energy transfer detection method and apparatus |
| US6117679A (en) * | 1994-02-17 | 2000-09-12 | Maxygen, Inc. | Methods for generating polynucleotides having desired characteristics by iterative selection and recombination |
| US5503977A (en) * | 1994-04-22 | 1996-04-02 | California Institute Of Technology | Split ubiquitin protein sensor |
| US5616475A (en) * | 1995-02-03 | 1997-04-01 | The Regents Of The University Of California | Human T-cell leukemia virus transcription modulators and screening assays |
| US5563039A (en) * | 1995-03-31 | 1996-10-08 | Tularik, Inc. | TNF receptor-associated intracellular signaling proteins and methods of use |
| EP1295952A3 (en) * | 1996-01-23 | 2003-07-09 | The Board of Trustees of The Leland Stanford Junior University | Methods for screening for transdominant effector peptides and RNA molecules |
| US6803188B1 (en) * | 1996-01-31 | 2004-10-12 | The Regents Of The University Of California | Tandem fluorescent protein constructs |
| US6022706A (en) * | 1997-04-09 | 2000-02-08 | Smithkline Beecham Corporation | Div1b |
| US6376257B1 (en) * | 1997-04-24 | 2002-04-23 | University Of Rochester | Detection by fret changes of ligand binding by GFP fusion proteins |
-
1997
- 1997-08-28 DE DE19737562A patent/DE19737562A1/en not_active Withdrawn
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1998
- 1998-08-28 CA CA002301633A patent/CA2301633A1/en not_active Abandoned
- 1998-08-28 AT AT98947468T patent/ATE210828T1/en not_active IP Right Cessation
- 1998-08-28 AU AU94375/98A patent/AU9437598A/en not_active Abandoned
- 1998-08-28 EP EP98947468A patent/EP1007968B1/en not_active Expired - Lifetime
- 1998-08-28 DE DE19881583T patent/DE19881583D2/en not_active Expired - Fee Related
- 1998-08-28 JP JP2000508983A patent/JP2001515208A/en active Pending
- 1998-08-28 DE DE59802465T patent/DE59802465D1/en not_active Expired - Fee Related
- 1998-08-28 WO PCT/EP1998/005458 patent/WO1999012033A2/en active IP Right Grant
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005050182A1 (en) * | 2003-11-19 | 2005-06-02 | Dimerix Biosciences Pty Ltd | Resonance energy transfer assay system for multi-component detection |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999012033A2 (en) | 1999-03-11 |
| EP1007968B1 (en) | 2001-12-12 |
| DE59802465D1 (en) | 2002-01-24 |
| EP1007968A2 (en) | 2000-06-14 |
| WO1999012033A3 (en) | 1999-06-10 |
| AU9437598A (en) | 1999-03-22 |
| DE19881583D2 (en) | 2001-06-21 |
| ATE210828T1 (en) | 2001-12-15 |
| DE19737562A1 (en) | 1999-05-06 |
| JP2001515208A (en) | 2001-09-18 |
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