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WO1998003676A1 - Procede pour detecter des effecteurs d'interactions proteine-proteine intracellulaires et/ou intercellulaires - Google Patents

Procede pour detecter des effecteurs d'interactions proteine-proteine intracellulaires et/ou intercellulaires Download PDF

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WO1998003676A1
WO1998003676A1 PCT/EP1997/003839 EP9703839W WO9803676A1 WO 1998003676 A1 WO1998003676 A1 WO 1998003676A1 EP 9703839 W EP9703839 W EP 9703839W WO 9803676 A1 WO9803676 A1 WO 9803676A1
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protein
ser
gin
hybrid
polypeptide
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Hanspaul Hagenmaier
Susanne Hagenmaier
Dieter Schrenk
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

  • the invention relates to a genetic detection method for effectors of intra- and / or intercellular protein-protein interactions. It is particularly applicable in the context of the so-called stress screening for effectors of intra- and / or intercellular protein-protein interactions.
  • the detection method according to the invention can also be used in the search for and isolation of unknown effectors of already known intra- and / or intercellular interactions at the protein level.
  • the invention also relates to nucleic acid sequences and analysis kits which are required for carrying out the detection method according to the invention.
  • Protein-protein interactions and the stable or transient protein complexes formed in this way are of crucial importance in the entire biosphere.
  • the formation, development and reproduction of organisms at the most diverse stages of development would not be conceivable without protein-protein interactions.
  • Protein-protein interactions are central cuts in biochemical processes such as DNA replication, transcription and translation, secretory processes, signal transduction, cell metabolism and cell cycle.
  • the ability of protein-protein interactions to be influenced by effectors can be explained, for example, using the cytoskeleton of eukaryotic cells.
  • the cytoskeleton not only contributes to the stability of eukaryotic cells, but also performs other important functions, such as the transport of vesi- none, changes in cell shape as well as the movement of cells.
  • This dynamic structure is formed by three classes of filamentous assemblies: the microfilament, the intermediate filament and the microtubule. Microfilaments are formed by polymerizing a protein called actin.
  • actin The protein-protein interaction of actin proteins required for the polymerization can be inhibited, for example, by the fungal alkaloid cytochalasin B. Cyto-chalasin interferes with the assembly of the actin filaments by binding (capping) to the free end of the last actin protein, which is important for further polymerization.
  • TCDD dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • TCDD and other dioxins are effective inducers of the transcription of a number of molecules at the molecular level
  • target genes encoding metabolic enzymes such as glutathione-S-transferase Ya, aldehyde dehydrogenase and quinone oxidoreductase (Landers, JP and Bunce, NJ (1991) Biochem. J. 276, 273 to 287).
  • Viruses are known to be pathogens for humans, animals and plants. They can cause relatively harmless illnesses like colds, but also life-threatening illnesses like polio or AIDS.
  • a virus is an intracellular parasite that can exist in two fundamentally different forms inside and outside a cell. The extracellular form is called virion or virus particle.
  • Virione consist of the viral genome, which with Proteins and sometimes associated with other chemical substances such as lipids. The proteins protect the genome from destruction in the extracellular space, allow entry into the host cell and often perform preliminary steps in the viral replication cycle. During the infection, the virion structure is largely or completely destroyed, so that the genome is freely available in the cell for transcription and replication.
  • virus particles which is made up of virus-coding proteins, the capsid, must be made up of one or a few different, repeating protein building blocks. This limitation is limited by the virus
  • the virion capsids have a basic blueprint with the following two properties: a) a narrow helix of undefined length with the genome in the center and b) a quasi-spherical icosaidric structure.
  • Virions of animal viruses can additionally differ due to the presence or absence of an outer membrane jacket. Virions that do not have this membrane envelope are called “naked”. Viruses that have this envelope, also called envelope, acquire it from the host's membrane using a two-step process called "budding”. First, viral glycoproteins are inserted into the membrane, which, by interacting with capsid proteins, coat the capsid with the membrane and release the finished virus particle to the outside.
  • capsid proteins associate with each other to form the capsid;
  • viral glycoproteins from the membrane of the infected cell interact with viral capsid proteins;
  • Virion proteins interact with cell surface antigens of the host cell to be infected.
  • the two-hybrid technique is a genetic method with which a protein-protein interaction is determined via the transcriptional activity of a hybrid protein complex produced in cell culture.
  • the technology is based on the modular structure of many site-specific transcription factors (transcription activators), consisting of a DNA binding domain and an activation domain.
  • transcription activators transcription activators
  • the two-hybrid technique is based on the observation that a direct covalent connection between these two domains is not required, but that a spatial proximity, caused by the interaction of any two other proteins, is sufficient to induce transcription.
  • it is necessary to construct two hybrid proteins and to express them together in a suitable host cell system, such as in yeast cells.
  • a sequence encoding a first protein involved in a protein-protein interaction is fused while maintaining the reading frame with the coding sequence of the DNA binding domain of the transcription factor, such as the yeast transcription factor GAL4, whereby the coding sequence for receives a first hybrid protein.
  • the coding sequence for a second hybrid protein is produced by fusing the coding sequence for the second protein involved in the interaction with the coding sequence for the activator domain of the transcription factor in the same reading frame.
  • a functional activator is generated in the host system after co-expression when the DNA binding domain and the activation domain come into spatial proximity due to the physical interactions between the two foreign protein components.
  • a reporter gene is expressed which is under the genetic control of an upstream activation sequence, such as GAL1 UAS, to which the DNA binding domain of the transcription factor binds.
  • the reconstitution of the function of the transcription factor caused by the interaction of the two foreign proteins thus causes the transcription of the reporter gene (such as lacZ or HIS 3).
  • the reporter gene such as lacZ or HIS 3
  • Its gene product, such as ⁇ -galactosidase can then be detected in a conventional manner.
  • the two-hybrid technique can also be used to detect those protein-protein interactions which are prevented in the presence of a known low molecular weight ligand (such as for example in the case of cyclosporin A) or only in the presence of the ligand (as in the case of rapamycin). Both works, however, have in common the search for a previously unknown protein binding partner. Without providing concrete examples, Chiu et al. that the two-hybrid technique could be used for tests with and the design of low molecular weight modulators of a protein-protein interaction, whereby the investigated modulators should have potential therapeutic value.
  • WO 95/26400 describes a method known as a reverse two-hybrid method which is intended to enable the search for molecules which inhibit protein-protein interactions.
  • the test system described therein is characterized in that a so-called signal inverter gene (or M relay gene ”) is used as an essential component in addition to the reporter gene. This measure is intended to prevent possible negative influences of cell toxins or translation inhibitors which could be present in a sample.
  • Screening procedures are usually one of them characterized in that the potential effector is not present as a pure substance but in a mixture with other compounds and in an extremely low concentration. This is particularly the case if new resources are examined for unknown effectors or if samples of the same type obtained in the same way are screened for a known effector.
  • the object of the present invention is therefore to provide a detection method for low-molecular effectors of intra- and / or intercellular protein-protein interactions, with the aid of which, in particular, mixtures of substances quickly and with high sensitivity to the presence of a known or unknown effector for a given protein protein - Interaction system can be examined.
  • the method according to the invention should be applicable in particular for so-called stress screening methods which can be used in the context of epidemiological studies.
  • the method according to the invention should also be suitable for carrying out so-called natural product screening methods in order to localize and subsequently analyze novel therapeutic active substances.
  • the object according to the invention is achieved by providing a genetic detection method for effectors of intra- and / or intercellular protein-protein interactions, wherein a) in a host cell in the presence of an analyte in which one suspects the effector function, two polypeptide hybrids A 1 A 2 and B- j ⁇ , whose domains A ⁇ and B- ⁇ together form a functional transcription-activator complex, if the domains A 2 and B 2 , which the intra- and / or intercellular interaction, as they for example takes place under physiological conditions, imitate, interact with one another, the interaction between the polypeptide domains A 2 and B 2 being observed only in the absence or presence of the effector function, ie at least one effector, in the analyte; and b) analyzed for expression of a reporter gene under the genetic control of an upstream transcription activation sequence to which the (in transcription activator complex formed in situ, expression of the reporter gene product indicating the presence or absence of the effector function in the an
  • Another object of the present invention relates to a method for isolating the effectors detected in this way and the new effectors of protein-protein interactions isolated using this method.
  • the invention relates also to nucleic acid sequences which encode the present invention used in the detection methods or hybrid proteins Polypeptidhybride A 1 A 2 and B ⁇ j. Finally, the invention relates to an analysis kit for carrying out the detection method according to the invention.
  • FIG. 1 shows the construction scheme of two expression vectors that can be used according to the invention before insertion of the coding sequence for the second hybrid protein component:
  • (A) shows the 5523 bp plasmid pGBT9, which contains the coding sequence for the DNA binding domain (bd) of the eukaryotic transcription activator GAL4.
  • (B) shows the 6659 bp plasmid pGAD424, which contains the coding sequence for the transcription activation domain (ad) of GAL4.
  • MSC stands for the multiple cloning site
  • p for the promoter
  • T for the telomerepression domain
  • Figure 2 shows the construction scheme of the expression vectors of Figure 1 after insertion of the coding sequence for the second hybrid protein component:
  • A illustrates the insertion of the coding sequence for the ARNT protein into the BamHI site of the MSC from pGBT9.
  • B shows the insertion of the coding sequence for the Ah protein into the Sall site of the MSC of pGAD424.
  • the detection method according to the invention offers the surprising advantage that effectors of a given protein-protein interaction can be detected qualitatively in an efficient manner, that is to say quickly and generally without separating the other low or high molecular weight components present in the analyte being examined.
  • a separation of the analyte or a further limitation of the effector is only necessary if it should be shown that more than one
  • effector of the protein-protein interaction in question is contained in the analyte or individual components of the analytes interfere with the test system.
  • the expert can easily determine the latter by means of a few control experiments.
  • the protein-protein interaction artificially induced in the test system according to the invention "imitates" or imitates the natural or physiological interaction insofar as at least the binding specificity of the interacting protein components observed under natural or physiological conditions is essentially retained in the test system.
  • the strength of the interaction in the test system can vary compared to the physiological process. It is therefore essential that the protein-protein interaction takes place functionally equivalent to the natural or physiological interaction under the test conditions.
  • the protein-protein interaction specified according to the present invention is of course not restricted to pure proteins or polypeptides.
  • the invention is also applicable to those interactions in which, for example, glycoproteins or lipoproteins are involved.
  • Test systems which can be used according to the invention can moreover not only be established as binding partners with complete proteins. Rather, functional equivalents, such as fragments, mutants and other derivatives, are also factors involved in the native protein-protein interaction.
  • Functional fragments can include partial sequences, such as certain domains, of the factor to which the binding function required for the interaction is located. Suitable fragments can be generated, for example, by genetic engineering or in the classical manner by enzymatic or chemical fragmentation.
  • Functional equivalents according to the invention also include derivatives of the binding partners generated by amino acid substitution, addition, insertion and / or deletion while maintaining the binding specificity, for example genetically or chemically.
  • the effector or modulator to be detected is preferably a low-molecular component which initiates, stimulates or inhibits the protein-protein interaction. So it can be a positive or negative effector of the interaction.
  • the analyte to be investigated according to the invention is preferably a mixture of substances, it being assumed that at least one of these substances has a positive or negative effector function on the predetermined protein-protein interaction.
  • This active substance is particularly preferably a low-molecular inorganic, preferably organic compound.
  • the molecular weight of the effector can be up to about 5000 g / mol, such as up to 2000 g / mol, and e.g. are in the range of about 10 to 1000 g / mol.
  • the analyte investigated according to the invention is selected, for example, from body fluid samples, such as whole blood, breast milk, cerebrospinal fluid, saliva and urine.
  • extracts or homogenates of procaryotic or eukaryotic cells such as bacterial homogenates, homogenates from plant cells or homogenates from lower eukaryotes, such as yeasts, or higher eukaryotes, such as animal or human cells, can be examined with the method according to the invention.
  • procaryotic or eukaryotic cells such as bacterial homogenates, homogenates from plant cells or homogenates from lower eukaryotes, such as yeasts, or higher eukaryotes, such as animal or human cells.
  • higher molecular components such as cell fragments, organo-le, or macromolecules, such as proteins and nucleic acids. separate.
  • Environmental analytical samples such as e.g. Water, air and soil samples, residues from industrial plants, e.g. liquid or solid special waste, filter dust from flue gas filter systems.
  • Food samples can also be analyzed according to the invention.
  • reaction mixtures of chemical syntheses if appropriate after suitable preparation, such as Exchange of solvent, concentration or dilution, to examine for effector function.
  • Reporter genes which can be used according to the invention for various host cell systems are known to the person skilled in the art from the specialist literature cited above, to which reference is hereby made. Examples are lacZ from E. coli and HIS3 and LEU2 from yeast. The only decisive factor is that the expression of the reporter gene allows the presence of an effector of the protein-protein interaction in the analyte examined in each case to be specifically indicated.
  • the predetermined intra- and / or intercellular protein-protein interaction is a process that occurs under physiological conditions in prokaryotic, e.g. bacterial cell systems, in eukaryotic cells, e.g. Yeast cells or human cells, or in viral microorganisms or in the interaction of different of these cell systems, e.g. an interaction of viral proteins with eukaryotic proteins takes place.
  • prokaryotic e.g. bacterial cell systems
  • eukaryotic cells e.g. Yeast cells or human cells
  • viral microorganisms e.g. an interaction of viral proteins with eukaryotic proteins takes place.
  • Test system in particular the host cells to be transformed, can mimic the protein-protein interaction under as physiological conditions as possible.
  • different types of protein-protein interactions can be used.
  • a first embodiment of the method according to the invention is based on such interactions that only occur when an effector is present in the analyte.
  • a po- Positive detection of the protein-protein interaction is therefore synonymous with positive effector detection.
  • the effector acts as a mediator or activator of the interaction.
  • Such a system is therefore particularly suitable for screening for mediators which initiate or stimulate a protein-protein interaction.
  • inhibitors of the interaction can also be detected.
  • a known mediator can be used in the test system and an analyte can be tested for the presence of an inhibitor of the mediator-dependent interaction, the inhibitor competing with the mediator, for example competitively, for binding to one of the protein components of the test system.
  • a second embodiment is based on interactions that are prevented in the presence of an effector.
  • a positive detection of the protein-protein interaction is then synonymous with a negative effector detection. If no protein-protein interaction is detectable, this indicates the presence of an inhibitor of the interaction in the analyte.
  • the methods according to the invention are preferably carried out in such a way that a first and a second nucleic acid sequence are introduced into a suitable prokaryotic or eukaryotic host cell system, the first nucleic acid sequence coding for the polypeptide hybrid A 1 A 2 , which codes the transcriptional Activation domain A j of the transcription factor, such as GAL4, and the polypeptide domain A 2 ; and the second nucleic acid sequence encodes the polypeptide hybrid B 1 B, which comprises the DNA binding domain B of a transcription factor, such as GAL4, and the polypeptide domain B 2 .
  • the two nucleic acid sequences can be present separately from one another or can be located on a single polynucleotide chain.
  • the sequences are introduced using known standard methods, preferably by transforming the host with a suitable vector.
  • (A 1 A 2 / B 1 B 2 ) essentially corresponds to the native transcription activator from which the hybrid protein domains A and B- ⁇ are derived.
  • Functional equivalents of these domains can also be used, which can be produced by amino acid addition, deletion and / or substitution while maintaining the characteristic properties, such as binding specificity and activation specificity, by genetic engineering or in another manner.
  • the analyte can be added to the test system before, with or after the introduction of the nucleic acid sequences.
  • the most suitable procedure for the particular analyte can easily be determined by a person skilled in the art on the basis of a few preliminary tests.
  • suitable solvents are unbuffered or buffered aqueous solutions, which may contain a solubilizer, such as e.g. an organic solvent, such as DMSO, or a cationic, anionic or nonionic surface-active compound, e.g. long chain fatty acids or salts thereof.
  • the host cell system can be applied or fixed to a solid or semi-solid support, such as, for example, poly beads or agar gels.
  • a solid or semi-solid support such as, for example, poly beads or agar gels.
  • the detection method according to the invention with host cells in suspended form. This is particularly suitable for screening procedures.
  • a fresh transformed host cell culture is prepared, the cell suspension is incubated with the analyte, if appropriate with gentle heating and / or gentle shaking or stirring.
  • the incubation time can vary over a wide range and can be, for example, 1 minute to 24 hours.
  • the detection of expression of the reporter gene is carried out.
  • effectors of cell-physiological interactions between proteins that can be detected in or between eukaryotic or prokaryotic cells, in virus particles or between virus particles articles and eukaryotic cells can in principle be a well-characterized, known substance or a substance not previously described in the specialist world.
  • 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mentioned as a preferred example of an effector which influences a cell-physiological process which takes place in eukaryotic cells.
  • TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • the cellular dioxin receptor system is a typical example of a signal-controlled dimerization of basic helix-loop-helix (bHLH) factors.
  • the cytosolic receptor molecule Before binding a Dioxin molecule, the cytosolic receptor molecule is composed of three subunits: the dioxin-binding Ah receptor and two molecules of the 90 kDa heat shock protein HSP90.
  • the HSP90 subunits have the function of keeping the Ah receptor in an inactive state in the absence of a ligand.
  • the two HSP90 molecules dissociate from the Ah receptor, which activates it and enables it to bind to the so-called Arnt protein.
  • the dioxin-laden complex thus formed is then capable of translocation from the cytosol into the cell nucleus.
  • XRE Xenobiotic responsible Element
  • a 2 is the first hybrid protein domain uses the Ah receptor or a binding fragment thereof and uses either HSP 90 or ds Arnt protein or a binding fragment of one of the two factors as the second hybrid protein domain B.
  • Nucleic acid and amino acid sequences of the Ah receptor, the HSP 90 and the Arnt protein are known from the prior art, so that the nucleic acid sequences required for carrying out the test for the production of the two polypeptide hybrids A 1 A 2 and B j ⁇ B- j can be easily provided.
  • two different test systems are conceivable for the dioxin detection according to the invention.
  • the binding of the dioxin molecule to the Ah receptor removes a protein-protein interaction, which has the consequence that the Expression of the reporter gene is prevented.
  • a binding of the dioxin molecule to the Ah receptor builds up a protein-protein interaction so that the expression of a reporter gene can be detected in the test system.
  • Two-hybrid test systems which have already been established for protein-protein binding tests, can be used to carry out the dioxin detection. For example, under the trade name MATCHMAKER Two Hybrid System from Clontech Laboratories Inc., Paolo
  • the system comprises two different expression vectors (pGBT9 and pGAD424), which genes for the DNA binding domain (pGBT9) and for the activation domain (pGAD424) of the yeast transcription - wear factor GAL4.
  • the pGBT vector is used to produce a first nucleic acid sequence which, for a hybrid, consists of a Gal4 DNA binding domain and Arnt protein.
  • a PCR polymerase chain reaction
  • H3 and H4 of the following sequence is to be constructed in vector:
  • H3 5'CGGGATCCCCGAAATGACATCAGATGT (SEQ ID NO: 3)
  • H4 5'CGGGATCCACCTGCTGGGCAGAAAAG (SEQ ID NO: 4)
  • the specific primers allow amplification of the Arnt reading frame with BamHI cleavage sites at its 5 'and 3' ends.
  • the ligation with the BamHI-treated pGBT9 vector takes place via the BamHI interfaces.
  • the pGAD424 expression vector of the matchmaker system is used to produce a second nucleic acid sequence which is used for a hybrid of the GAL4 activation domain and the Ah receptor.
  • the reading frame of the Ah receptor is based on the Ah cDNA with the help of the specific oligonucleotides Hl and H2
  • H2 5'AGGTCGACTTATGAGCAGCGGCGCCAACAT (SEQ ID NO: 2)
  • oligonucleotides provide the reading frame of the Ah receptor with Sall cleavage sites.
  • the Ah sequence modified in this way can be ligated with the Sall-cut pGAD424 vector.
  • the two hybrid vectors pGBT9-Arnt and pGAD424-Ah produced in this way are first transformed, propagated and purified in E. coli. If the isolated plasmids show the desired constructs, pGBT9-Arnt and pGAD424-Ah are cotransformed in SFY526 yeast host cells. If dioxin is present in the growth medium, the result is a dimerization of the two expressed fusion proteins. The dimerization of the two GAL4 domains in the two hybrid proteins leads to the activation of the transcription of the reporter gene ⁇ -galactosidase, the formation of which is verified in the usual way by means of a color reaction. will be shown.
  • the detection sensitivity for 2,3,7,8-TCDD per batch is in the lower femtomol to upper atomol range and thus in the range of the detection limit of the most sensitive mass spectrometers currently available on the market.
  • HSP90 protein not only shows a regulatory effect on the Ah receptor, the natural ligands of which are unknown, but can be detected using the screening method according to the invention.
  • HSP90 also acts as a repressor for steroid receptors such as the estrogen and androgen receptors. Since more and more xenobiotics are identified for which an estrogen or androgen-like effect is suspected, a detection method according to the invention would also be suitable for screening for such substances.
  • the effector to be identified should have an inhibitory effect on an interacting hybrid protein pair, comprising the hybrid proteins steroid receptor-Al and HSP90-B1, where AI and B1 together should form the functional transcription activator complex.
  • a detection method for effectors of viral protein-protein interactions is provided. This can be carried out in complete analogy to the dioxin test described above.
  • interactions between the poliovirus capsid proteins VP1 and VP3 and between two pl ⁇ -capsid proteins or two p24-capsid proteins from HIV-1 are tested.
  • Examples of the composition in a hybrid construct A 1 A 2 and B 1 B which can be used for the detection of viral protein-protein interactions according to the invention are given in Table 1 below. Table 1
  • a l DNA binding domain
  • a 2 first viral target protein
  • Bl activator domain
  • VP1 and VP3 can also be interchanged in the above hybrids.
  • the coding and provided with suitable restriction cleavage DNA sequences for the viral hybrid protein components A2 and B2 can be prepared and inserted into the vectors pGBT9 and pGAD424 described above.
  • the two-hybrid test for detecting an effector of the viral protein-protein interaction is then carried out in analogy to the dioxin test described above.
  • This test system can also be applied to all other protein-protein interactions involving viral proteins.
  • Viruses which, in addition to the protein capsid, also have an envelope with glycoproteins can also be used as the basis for a method according to the invention.
  • the glycoproteins of the envelope interact with the capsid proteins. This interaction is necessary to equip the virus with the envelope structure and at the same time to remove it from the cell.
  • the Semliki Forest virus shows two spike glycoproteins El and E2, which, like many other viral envelope glycoproteins, consist of three domains: one external, one transmembrane and one internal domain. The internal, C-terminal domain interacts with the capsid proteins.
  • a two-hybrid test for the detection of effectors of this interaction can be established in such a way that the coding sequence for the capsid protein and the envelope glycoprotein, or preferably the DNA sequence of interacting fragments of these two proteins in the Vectors described above inserted and the genetic information contained on the vectors expressed in the presence of an analyte.
  • Envelope proteins thus represent another preferred embodiment.
  • test systems which are based on defined protein-protein interactions with the participation of eukaryotic, prokaryotic, and / or viral proteins.
  • the invention is not restricted to the use of the specific vectors and host cells described above, but rather all other comparable test systems, such as e.g. the expression vectors and host cells described so far in the specialist literature can be used.
  • the present invention further relates to a method for isolating new effectors of intra- and / or intercellular protein-protein interactions, in which a) an analyte is subjected to a detection method as defined above; b) with positive proof of an effector function in the
  • Analytes are limited using conventional preparative methods, if necessary by further separation of the analyte, and the effector, if necessary with the aid of the detection method as defined above, or in another way, such as by spectroscopy, high-pressure liquid chromatography, thin-layer chromatography, localized and isolated using conventional preparative methods such as chromatography.
  • the invention further relates to nucleic acid sequences, such as, for example, DNA, RNA or cDNA sequences, which code for polypeptide hybrids which contain polypeptide domains as defined above or functional equivalents thereof.
  • nucleic acid sequences are the sequences coding for the polypeptide hybrids GAL4 binding domain / Arnt and GAL4 activation domain / Ah.
  • the invention also relates to an analysis kit for carrying out one of the detection methods described above.
  • the analysis kit according to the invention is characterized in that it comprises, preferably in separate compartments: a) a first coding nucleic acid sequence, such as, for example, a transformation vector coding for a polypeptide hybrid A 1 A 2 , b) a second nucleic acid sequence, such as for example a transformation vector coding for a polypeptide hybrid B 1 B 2 and optionally c) a host cell culture which contains the genetic information for a reporter gene which is under the genetic control of an upstream transcription activation sequence to which, after introduction of the first and second nucleic acid sequence, for example by transformation, the transcription-activator complex A- j ⁇ binds into the host cell if the hybrid protein domains A 2 and B 2 interact with one another in the presence or in the absence of an effector .
  • a first coding nucleic acid sequence such as, for example, a transformation vector coding for a polypeptide hybrid A 1 A 2
  • b) a second nucleic acid sequence such as for example a transformation vector
  • first and second nucleic acid sequences can also be arranged on a single vector.
  • the vectors used according to the invention usually contain the coding sequences under the control of customary regulatory sequences known to the person skilled in the art which control the expression of the hybrid proteins in the respective host.
  • the present invention will now be explained in more detail with reference to the following exemplary embodiments, which relate to the establishment of a dioxin screening system. example 1
  • oligonucleotide Hl 5'AGGTCGACACTCTGCACCTTGCTTAGGAAT oligonucleotide H2: 5'AGGTCGACTTATGAGCAGCGGCGCCAACAT oligonucleotide H3: 5 'CGGGATCCCCGAAATGACATCAGATGGGATCCAGAAGATGCT 5GAGGACACAQGGGAGGACACA
  • the PCR protocol was carried out in a slightly modified form in accordance with the information from Perkin-Elmer-Cetus (protocol for DNA amplification). The following reaction mixture was pipetted for the amplification of a DNA fragment using PCR:
  • the reaction was carried out in a final volume of 100 ⁇ l in siliconized, autoclaved reaction vessels.
  • the PCR was carried out in the DNA thermal cycler for 1 min at 95 ° C, 3 min at 60 ° C and 2.5 min at 72 ° C.
  • the 72 ° C step was extended by 2 s per cycle.
  • the PCR comprised a total of 30 cycles.
  • the PCR was started with a "hot start” (at the beginning 5 minutes at 95 ° C.) and ended after 10 minutes at 72 ° C.
  • the plasmids pGAD424 and pGBT9 (Fig. 1 (A) and (B) are available from Clontech Laboratories, Inc., Palo Alto, CA, USA.
  • Ah cDNA and Arnt cDNA can be isolated in a conventional manner from cells expressing Ah and Arnt protein.
  • the cDNA cloning and DNA and amino acid sequence of murine Ah protein are described, for example, by Erna, M. et al. In Biochem. Biophys. Res. Communications (1992), Vol. 184, 246-253, the content of which is hereby expressly incorporated by reference.
  • the murine Ah protein is a polypeptide of 805 amino acids with a calculated molecular weight of 90380 daltons.
  • Arnt protein contains 789 amino acids and has a calculated molecular weight of 86637 daltons.
  • the coding sequence of the Ah receptor was amplified using the PCR method using the specific oligonucleotides H1 and H2, starting from the Ah cDNA matrix.
  • the synthetic oligonucleotides were constructed so that Sall interfaces were introduced into the amplified Ah fragment at the 3 'and 5' ends.
  • the purified PCR Ah fragment was subjected to a Sall restriction digest and ligated into the pGAD424 vector, which was also cut with Sall (FIG. 2 (B)).
  • the clones obtained after transformation into the E. coli strain MC4lrF '(selection for amp resistance) were examined with regard to their plasmids.
  • the coding Sequence of the Arnt receptor (SEQ ID NO: 6) amplified.
  • the synthetic oligonucleotides were constructed so that BamHI cleavage sites were introduced into the amplified Arnt fragment at the 3 'and 5' ends.
  • the purified PCR-Arnt fragment was subjected to a Ba HI restriction digest and ligated into the pGBT9 vector which had also been cut with BamHI (FIG. 2 (A)). The clones obtained after transformation into the E.
  • coli strain MC41rF '(selection for amp resistance) were examined with regard to their plasmids. Using an EcoRI restriction digest, it was possible to determine which bacterial clones contained the desired construct pGBT9-Arnt. The corresponding bacterial clone was expanded and the plasmid pGBT9 Arnt DNA was isolated.
  • the QIAGEN plasmid kit in combination with QIAGEN-Tip-200 columns was used for the plasmid isolation from transformed E. coli cells. The plasmid DNA was isolated according to the manufacturer's instructions. A 150 ml overnight culture of the transformed E. coli cells was used for plasmid isolation. The plasmid yield of isolation for pGBT9-Arnt was between 0.5 and 1 mg / ml.
  • 20 ml of YPD medium were inoculated with a single yeast colony and shaken at 30 ° C. overnight.
  • the 20 ml overnight culture should have reached the stationary phase after this time, ie show an OD> 1.5.
  • the overnight culture was used to inoculate 300 ml of fresh YPD medium; this 300 ml culture with an OD 60 o of 0.2-0.3 was shaken at 30 ° C. for 3 hours.
  • the cells were centrifuged off, the pellet was washed twice in 50 ml of sterile, double-distilled water and in 1.5 ml of fresh, sterile IxTE - " iAC buffer (prepared immediately before use from 10XTE buffer (0.1 M Tris HC1, lO M EDTA, pH 7.5) and lOxLiAC buffer (IM lithium acetate, pH 7.5)).
  • the competent yeast cells were used immediately for the transformation.
  • 0.1 ml of competent yeast cells were pipetted into 0.1 ⁇ g of plasmid DNA and 100 ⁇ g of carrier DNA (“herring testes carrier DNA”) and 0.6 ml of PEG / LiAc solution (40% PEG 4000, IxTE, lx LiAC, fresh) manufactured) .
  • carrier DNA herein carrier DNA
  • PEG / LiAc solution 50% PEG 4000, IxTE, lx LiAC, fresh
  • the mixture was mixed well and shaken at 30 ° C. for 30 minutes. After the addition of 70 ⁇ l DMSO, there was a 15 min heat shock at 42 * C.
  • the cells were then cooled on ice, centrifuged and the pellet resuspended in 0.5 ml TE buffer. 0.25 ml of the transformed cells were plated out on SD plates (-Leu, -Trp) and the plates were incubated for 2-4 days at 30 ° C. until the first colonies appeared.
  • pLAM 5 encodes a hybrid of GAL4 binding domain and human Lamin C
  • pTDl codes for a hybrid of GAL4 activator domain and SV40 large T antigen
  • pCLl codes for a full-length GAL 4.
  • the plasmids pLAM5 ', pTDl and pCLl are also available from Clontech Laboratories Inc.
  • the SSX indicator plates (Chien et al. 1991) have the following composition:
  • the SSX plates contain the substance X-Gal, which is converted into a blue dye after interaction of the fusion proteins. While the control yeasts (pLAM5 '/ P CL -L) showed a clear blue color after growth (o / n) on SSX plates, this was not the case with the other plasmid combinations.
  • the pGAD424-Ah / pGBT9 type SFY5426 yeasts were placed on the SSX plates after placing 2, 3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) containing pads (TCDD was applied to the pads in 10% DMSO) Recognize specific, concentration-dependent blue staining in the area of the contact areas, but not the control yeasts (pGAD424 / pGBT9-Arnt; pGAD424-Ah / pGBT9, pLAM 5 '/ pTDl), the detection limit for 2,3,7,8-TCDD was in the range of 0.1 ng per pad.
  • the yeast strain SFY526 cotransformed with pGAD424-Ah and pGBT9-Arnt was cultivated overnight at 30 'C in SD medium -Leu, -Trp.
  • the cotransformations a) to d) mentioned under 1.3.3 served as a control.
  • the enzyme reaction was started by adding 0.16 ml of ONPG solution (4 mg / ml o-nitrophenyl- ⁇ -D-galactopyranoside in Z buffer).
  • ONPG solution 4 mg / ml o-nitrophenyl- ⁇ -D-galactopyranoside in Z buffer.
  • the release of ONP is concentration-dependent in the range from 50 to 500 fg 2,3,7,8-TCDD.
  • the liquid test turned out to be very sensitive for the detection of the dioxin ligand.
  • the controls which had been incubated without dioxin only with the solvent 5% DMSO
  • the sample was divided into 3 aliquots, the solvent was removed in a stream of nitrogen and the residue was taken up in the 3 vials with 10 ⁇ l, 30 ⁇ l and 100 ⁇ l 5% DMSO in water. 5 ⁇ l of the respective sample was introduced into liquid culture in the test described under 1.4.2 above.
  • TCCCTTCCCT CACCCCTGAC ATGTACCCCC TTTCCCTTCT GGCTGTTCCC CTGCTCTGTT 2583 GCCTCCTAAG GTAACATTTA TAAAAAAAAA AAA 2616

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Abstract

L'invention concerne un procédé génétique pour détecter des effecteurs d'interactions protéine-protéine intracellulaires et/ou intercellulaires, un procédé pour isoler de nouveaux effecteurs, des séquences d'acide nucléique codant pour les protéines hydrides utilisées dans le procédé de détection selon l'invention, ainsi qu'un kit d'analyse pour mettre en oeuvre ledit procédé de détection.
PCT/EP1997/003839 1996-07-17 1997-07-17 Procede pour detecter des effecteurs d'interactions proteine-proteine intracellulaires et/ou intercellulaires Ceased WO1998003676A1 (fr)

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EP0921192A1 (fr) * 1997-12-03 1999-06-09 Leadd B.V. Molecules interagissant avec l'apoptine
WO2000046406A2 (fr) * 1999-02-05 2000-08-10 Alphagene, Inc. Reseaux d'interaction proteomique

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US5378822A (en) * 1993-04-08 1995-01-03 Wisconsin Alumni Research Nucleic acids encoding murine and human Ah receptors
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US5378822A (en) * 1993-04-08 1995-01-03 Wisconsin Alumni Research Nucleic acids encoding murine and human Ah receptors
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HOFFMAN E C ET AL: "CLONING OF A FACTOR REQUIRED FOR ACTIVITY OF THE AH (DIOXIN) RECEPTOR", SCIENCE, vol. 252, 17 May 1991 (1991-05-17), pages 954 - 958, XP000674442 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002037077A3 (fr) * 2000-11-06 2002-11-07 Medexbio Co Techniques de criblage reposant sur un systeme d'analyse de regulation transcriptionnelle au moyen de lignees cellulaires transformees

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