WO2001025420A2 - Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis - Google Patents
Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis Download PDFInfo
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- WO2001025420A2 WO2001025420A2 PCT/US2000/027677 US0027677W WO0125420A2 WO 2001025420 A2 WO2001025420 A2 WO 2001025420A2 US 0027677 W US0027677 W US 0027677W WO 0125420 A2 WO0125420 A2 WO 0125420A2
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- Genetically-based interaction systems are commonly used in scientific research and in commercial and therapeutic applications derived from that research. Current genetically-based interaction systems are severely limited by a fixed level of interaction sensitivity which is either completely "on” or completely “off (Fields and Song, 1989; Bartel et al., 1993; Gyuris et al., 1993; Mendelsohn and Brent, 1994; Phizicky and Fields, 1995; Bai and Elledge, 1997; Brachmann and Boeke, 1997; Finley and Brent, 1997; Young, 1998). This creates problems related to both the detection of numerous biologically irrelevant interactions, as well as a failure to detect relevant biological interactions.
- the host cells were additionally transformed with a second hybrid protein consisting of a transcriptional activation domain and a second polypeptide of stable interaction with the first protein fragment. Finally, the cells were also transformed with a lacZ reporter gene containing at least one DNA binding sequence for the DNA binding domain of the first hybrid protein and capable of being transcribed at increased and detectable levels when the transcriptional activation domain of the second hybrid protein was in close proximity. Field and Song demonstrated that when the two hybrid proteins were expressed, levels of the LacZ reporter protein dramatically increased in the host cell.
- Genetic molecular detection systems following the original Fields two-hybrid system also usually include the additional feature of genetic selection (Fields and Song, 1989). Selection allows the detection of an interaction by choosing the phenotype of survival; cells containing proteins that do not interact strongly enough or at all are unable to grow, and are no longer considered.
- the current methods of selection are limited to an "all or nothing" auxotrophic nutrient, antibiotic selection or other means of affecting survival (Fields and Song, 1989; Gyuris et al., 1993; Bai and Elledge, 1997). Selection yields a great advantage for the various detection systems, since cells containing potentially irrelevant pairs of candidate interacting molecules are eliminated without intervention from the scientist or other automated analysis.
- a feature of current detection systems is the capacity to turn the detection of protein interactions on or off completely by providing for the expression or lack of expression of the two-hybrid library fusion under standard nutrient conditions.
- Gyuris et al. (1993) found that by being able to express one of the two hybrid proteins at high levels or by being able to limit expression of one such protein completely, it was possible to show in vivo that the presence of both of the hybrid proteins were necessary for activation of the reporter gene; in other words, they added a switch enabling on or off control of one of the interacting components. This control is useful and exerts its effects by modulating reporter activity, but it does not provide for the continuous adjustability of the sensitivity of a two-hybrid protein interaction system.
- Gyuris system further demonstrates the limitation of the prior art: it is either on or off, above or below the same detection threshold set by the reporters chosen when the system was constructed.
- the level of reporter gene expression that will result from any given molecule-molecule interaction in a two-hybrid system is uniform for those molecules used in combination with that reporter.
- the Brent lab first demonstrated this in experiments using a traditional two-hybrid protein-protein interaction system. The experiments showed that output of the quantitative lacZ reporter was directly proportional to the independently determined strength (or Kd) of the protein-protein interaction for the protein fragments used in the hybrid proteins. If the two proteins interacted strongly in vitro, they gave robust expression from the two-hybrid reporters and vice versa. Therefore, they also demonstrated that the output of a given reporter is constant for a given pair of interacting proteins. This is now generally accepted, since many publications of genetic molecular interactions include the quantitative reporter output from the interaction system as a relative indication of the strength of the interaction itself (Edwards et al., 1997).
- the present invention yields surprising and unexpected advantages relative to earlier systems in providing for adjustability of the sensitivity of such detection systems.
- the present invention comprises an improved two-hybrid or three-hybrid detection method and a kit utilizing this method.
- the method of the current invention may be used with any conventional two-hybrid or three-hybrid methods, including inhibition or competition two- hybrid methods, as well as any future variations of those methods.
- the sensitivity of a detectable reporter gene in a host cell is continuously adjustable by altering the relative or absolute amounts of interacting molecules provided to the host cell.
- the method may be used to detect interactions between any types of molecules including, but not limited to, proteins, polypeptides, DNA molecules, RNA molecules, pharmaceutical agents, other biological or chemical agents, and other small molecules or macromolecules.
- the method may be used to detect interactions in both prokaryotic and eukaryotic organisms or cells.
- the molecular interactions may occur at various locations, including, but not limited to, extracellular regions, the cell membrane, the cytoplasm, the nuclear membrane, the nucleus, and other intracellular regions.
- the first chimeric gene and the second chimeric gene are introduced into the host cell.
- the host cell is then subjected to conditions under which a first hybrid protein and a second hybrid protein are expressed in at least sufficient quantities for the detectable reporter gene within the host cell to be activated.
- the first chimeric gene contains a first exogenously activatable promoter and a sequence encoding the first hybrid protein.
- the first hybrid protein contains a DNA binding domain capable of binding near the reporter gene and a first interacting polypeptide(bait).
- the second chimeric gene contains a second exogenously activatable promoter and a sequence encoding a second hybrid protein.
- the second hybrid protein contains a transcriptional activation domain capable of inducing or increasing transcription of the reporter gene and a second interacting polypeptide(prey). This second polypeptide may be derived from a library.
- the sensitivity of the reporter gene may be altered by adding a first and/or second exogenous activator and thus, altering the relative or absolute amounts of the first and/or second hybrid proteins. These alterations affect the activity and thus sensitivity of the reporter gene.
- the sensitivity of this activation may be decreased by adding a first exogenous activator capable of activating the first exogenous promoter. This results in increased production of the first hybrid protein and raises its level in the host cell relative to the level of the second hybrid protein. Thus, after this increase, more DNA binding sites of the reporter gene are occupied by the first hybrid proteins for which there is not second hybrid protein available for interaction. Therefore, less of the reporter genes are activated or activation is weaker.
- the sensitivity of the reporter activation may be increased by adding a second exogenous activator capable of activating the second exogenous promoter. This results in increased production of the second hybrid protein and raises its level in the host cell relative to the level of the first hybrid protein. Thus, after this increase, more of the DNA binding sites of the reporter gene are occupied by a first hybrid protein that is additionally interacting with a second hybrid protein. Therefore, more of the reporter genes are activated or activation is stronger. Subsequent to the hybrid protein expressions, detectable reporter gene expression is measured and compared to the amount of expression in the absence of any interaction between the first test protein and the second test protein.
- kits utilizing the method of this invention may also be prepared.
- the kit may comprise any host cell described above, any first or second chimeric genes described above, or any combination thereof.
- the kit may also contain the first and second exogenous activators and also chemicals or assays for detecting the detectable reporter gene product.
- Figure 1 shows the Original Two-hybrid system relies on the reconstitution of a functional transcription factor to report the interaction of two proteins, depicted as X and Y.
- Fields two-hybrid system uses the DNA-binding domain and activation domain from the Gal4p transcription factor (figure adapted from Fields and Song, 1989).
- Figure 2 shows the Novel Library vector described herein is a shuttle vector containing ampicillin and the colEl origin of replication for selection in E.coli as well as TRP1 and the 2 micron origin of replication for selection in yeast.
- Unknown cDNAs are fused to the Gal4p activation domain, and continuously variable expression is obtained by the induction of GRE upstream activating element(s) attached to CYC1 promoter.
- a known cDNA is fused to the Gal4p DNA binding domain, and continuously variable expression is obtained by the induction of ERE element(s) attached to CYC1 promoter.
- GRE Glucocortocoid Response Element
- CYClp CYC1 promoter from yeast
- Gal4pAD Gal4 activat. domain (768-881)
- AdhT Alcohol dehydrogenase terminator.
- Figure 4 shows Continuously Dose Responsive Expression of proteins fused to Gal4pBD in the "Bait Vector" in yHYBOOl strain. Strain yHYBOOl with the bait vector is grown in selective minimal media with varying concentrations of estradiol. The bait vector contains the
- Gal4pBD fused to the marker lacZ.
- ⁇ -gal expression assays are performed three times per estradiol concentration; the data represents an averaging of three assays per sample. Growth was overnight and strains were at OD 600 ca. 0.8 when assayed. Strain yHYBOOl is described in the text, ⁇ -gal expression assays are described in Guarente (1983).
- Figure 5 shows Continuously Dose Responsive Expression of proteins fused to Gal4pAD in the "Bait Vector" in yHYBOOl strain.
- Strain yHYBOOl with the library vector is grown in selective minimal media with varying concentrations of dexamethasone.
- the bait vector contains the Gal4pAD fused to the marker lacZ.
- ⁇ -gal expression assays are performed three times per dexamethasone concentration; the data represents an averaging of three assays per sample. Growth was overnight and strains were at OD 600 ca. 0.8 when assayed.
- Strain yHYBOOl is described in the text, ⁇ -gal expression assays are described in Guarente (1983).
- Figure 6 shows the Principle of Variable Reporter Output with changes in relative concentration of interactors in a novel molecular genetic interaction detection system.
- the number of bars represent relative levels of library fusion protein and bait fusion protein present in the cell.
- At equilibrium only a fraction of the fusion proteins will be physically paired at any given time, representing the Kd of the interaction (in this example, we assume 50% are bound at a given time.)
- a medium sensitivity assay results when both fusion proteins are present in roughly equal amounts. At equilibrium, half of them are interacting, resulting in an output that is Vi of the theoretical maximum from the reporter. This is true since half of the DNA-binding domain fusions at the reporter will not be paired to an activating library fusion.
- This invention comprises novel compositions, methods and uses for continuously and/or discontinuously adjusting the sensitivity of genetic detection systems to enable significantly improved detection and analysis of molecular interactions.
- the molecular interactions include those at extracellular, membrane and intracellular sites, and include but are not limited to protein- protein interactions, protein-DNA interactions, protein-RNA and protein-small molecule interactions in eukaryotic and prokaryotic organisms.
- the method of the present invention involves changing the relative quantity of each macromolecular or small molecular component provided within the system, such that the absolute or relative amounts of actual interacting pairs changes within the system.
- the relative amounts of interacting molecules particularly the molecule bound to the detectable reporter gene
- the output of the system via reporter gene expression is also altered.
- the host cell may be provided with a detectable reporter gene.
- This reporter gene may be provided before or after interacting molecules or other components are provided. However, the reporter gene is preferably provided first so that host cells that do not contain the reporter gene may be eliminated before interacting molecules or other components are introduced.
- the reporter gene may be provided through any method of gene transfer currently known or later developed. In a preferred embodiment, the reporter gene is provided through electroporation of the host cell.
- the reporter gene may also be provided in any form capable of transfer to the host cell using the selected transfer method. For instance, it may be provided as a portion of a plasmid.
- any detectable reporter gene that may be activated by an interaction of the interacting molecules is appropriate for this method.
- the reporter gene may produce a detectable reporter protein or other detectable gene product.
- the method of this invention can function with any detectable reporter gene because the method involves changing the amounts of the interacting macromolecules themselves. If there are more interaction to report, any reporter gene will report this as a relatively stronger activation of the reporter gene. If there are relatively fewer interactions to report, any reporter system will report this as a relatively weaker output.
- the reporter gene comprises at least one DNA binding site capable of interaction with a polypeptide including a DNA binding domain or with another DNA binding molecule, such as a small molecule or pharmaceutical agent.
- This DNA binding site is located such that if a first interacting molecule binds to the site and additionally interacts with a second interacting molecule, the transcriptional activation domain of the second molecule will be able to activate transcription of the reporter gene.
- a first interacting molecule which may be a macromolecule or small molecule into a host cell or its extracellular region. This molecule should contain a polypeptide containing a DNA binding domain or it should contain another molecular region capable of binding DNA.
- This first interacting molecule may be a protein, a DNA, a RNA, or a pharmaceutical agent or any other molecule that contains or may be bound to a molecule containing a DNA binding region or domain.
- the molecule is a protein, a DNA, or a RNA.
- the macromolecule is provided by introducing into the host cell a first chimeric gene capable of being transcribed in the host cell.
- This first chimeric gene may include a first exogenously activatable promoter, a sequence coding for a polypeptide, DNA, or RNA containing a DNA binding region, and a sequence coding for the first interacting macromolecule.
- the first chimeric gene comprises a first exogenously activatable promoter and a first hybrid protein.
- This first hybrid protein comprises a DNA binding polypeptide and a first interacting polypeptide (bait) capable of interacting with at least one second interacting polypeptide (prey).
- a second interacting macromolecule or small molecule is also introduced into the host cell or its extracellular region.
- This molecule should contain a transcriptional activation domain.
- This transcriptional activation domain may be a polypeptide or a region of another molecule capable of activating transcription such as a region of a pharmaceutical agent or a nucleic acid.
- this second molecule may be a protein, a DNA, a RNA, a pharmaceutical agent, or any other molecule meeting the requirements stated above. In a more preferred embodiment it is a protein, a DNA, or a RNA.
- the macromolecule may be produced in the host cell by introducing a second chimeric gene capable of being transcribed in the host cell.
- This second chimeric gene may include a second exogenously activatable promoter, a sequence coding for a transcriptional activation domain, and a sequence coding for the second macromolecule.
- the chimeric gene may contain a second exogenously activatable promoter and a second hybrid protein. This second hybrid protein may contain a polypeptide containing a transcriptional activation domain and the second interacting polypeptide (prey).
- the sensitivity of the detectable reporter gene in a host cell is continuously adjustable by altering the relative or absolute amounts of the first and/or second interacting molecules provided to the host cell.
- the host cell itself has the capacity to regulate the absolute of relative levels of the first or second molecules. As indicated in the preferred embodiments described above, this may be accomplished by introducing chimeric genes encoding the first and second macromolecules and containing first and second exogenously activatable promoters. These exogenously activatable promoters may be activated by exogenous activators.
- the activator is a natural or synthetic, metabolically active or inactive steroid, steroid analogue or mimic and the promoter induces transcription in response to the activator.
- the relative or absolute amount of at least one of the hybrid proteins may then be controlled in a manner responsive to the dose of one or more of these activators, its analog, or its antagonist.
- the promoters will be different for each gene and will be activated by different molecules. By regulating the relative levels of the first and second interacting molecules, it is possible to alter the sensitivity of the reporter.
- the system is flooded with one component, usually the second molecule, it is possible to drive the system towards interaction of a first molecule bound to the reporter with a second molecule such that reporter activity is increased.
- a first molecule bound to the reporter with a second molecule such that reporter activity is increased.
- every first molecule binding the reporter is involved in an interaction with a second molecule, and therefore the reporter is activated more often or more strongly.
- the cells may additionally be provided with other macromolecules or small molecules that mediate or interfere with the interaction between the first and second interacting molecules.
- a third macromolecule or small molecule may be provided that facilitates or is required for the interaction of the first and second molecules.
- This third molecule will most commonly be a protein. It may exert its effect by stabilizing the interaction between the first and second molecules or by forming a connection between them when they otherwise would not interact.
- this third molecule may also interfere with the interaction of the first two.
- the third molecule may contain a polypeptide that is identical or similar to the bait or prey polypeptides.
- the third molecule will interfere with the ability of the bait and prey to interact.
- This variation of the two- hybrid assay is commonly known as a inhibition or competition two-hybrid assay. It is especially amenable to the method of the present invention because such assays do not currently provide precise results.
- inhibition two-hybrid assays would benefit greatly from the present invention because relative amounts of the bait and prey polypeptides greatly influence the ability of the third molecule to inhibit the bait-prey interaction and thus, the sensitivity of the reporter system.
- the techniques for fine-tuning and varying the amounts of the hybrid proteins of this method might also be applied to regulate the relative or absolute amount of a third inhibition polypeptide in an inhibition two-hybrid assay.
- the interaction between the first and second interacting molecules may take place and be detected anywhere within the cell. For instance, it may occur in the nucleus, in the cytoplasm, at or in the membrane, or in an organelle.
- the system would be expected to work similarly in prokaryotes and eukaryotes, including bacteria, yeast, plant, arthropod, and mammalian cells.
- the method is applied to a genetic molecular interaction detection system.
- Regulation of the amounts of hybrid proteins is accomplished by using compositions comprising alternate promoters for different intrinsic levels of expression of a downstream hybrid protein or molecule. These promoters may be derived from natural or synthetic, yeast or non-yeast sources. Regulation may be accomplished by several methods, including, but not limited to: (a) changing the promoter upstream of a hybrid protein, for instance a hybrid in which the second interacting (prey) polypeptide is derived from a library, to give different levels of expression, as further exemplified below, using a GAL1/10 promoter, CYC1 promoter, or ADH1 promoter, which exhibit different levels of expression
- regulation of hybrid protein amounts is accomplished by using a single promoter, for example, GALl/10, CYCl, ADHl or other natural or synthetic yeast or non-yeast promoters in combination with different upstream enhancer sequences from yeast or non-yeast sources.
- a single promoter for example, GALl/10, CYCl, ADHl or other natural or synthetic yeast or non-yeast promoters in combination with different upstream enhancer sequences from yeast or non-yeast sources.
- the method of the present invention discloses surprising and unexpected results applicable to all known genetic systems for detecting and analyzing protein interactions with other proteins and with any other classes of molecules. It is clearly and categorically distinguished from the prior art, based on its sensitivity being continuously adjustable; that is, the sensitivity may be adjusted on a plurally stepped dose-responsive basis. This includes, in one preferred embodiment, pharmacologically modifying the transcription or expression of the fusion protein or molecule and detecting the various reporter gene expression levels in a single screen.
- This embodiment of the present invention gives major technical advantages including, but not limited to: (a) detecting and analyzing interactions of various strengths, without any prior knowledge of even the range of such interaction strengths; (b) avoidance of biologically non- relevant interactions; (c) the detection of potentially very important but currently systematically undetected, weak interactions; and (d) the potential for actually quantifying the in vivo strength of intermolecular binding, as characteristically defined by dissociation constant (Kd) (Estojak et al., 1995).
- Kd dissociation constant
- an extracellular ligand binds and modulates the activity of a specific transmembrane receptor to effect a dose-responsive change in expression or activity of one or more interacting molecules.
- extracellular ligands include but are not limited to growth factors, cytokines, hormones, synthetic agents and biopharmaceuticals and their cellular receptors (Mercurio and Manning, 1999; Baldwin, 1996; Mohal and Steinberg, 1999).
- an intracellular ligand interacts either cytoplasmically or within the nucleus to modulate the expression or activity of the interacting molecules.
- intracellular ligands include but are not limited to small molecular pharmaceutical agents and modulators, including but not limited to antimicrobial agents, anti- tumor agents, nucleic acid-binding agents, cytoskeletal active agents, chelating agents, inducers, co-repressors, and agents affecting intracellular trafficking, localization and protection or degradation (Schena et al, 1991 ; Rossi and Blau, 1998).
- a membrane-active agent interacts to modulate the level of cellular activation or response potential.
- examples of such an agent include but are not limited to ionophores, amphoteric and hydrophobic lipid-active agents and detergents, various anesthetics and solvents, transmembrane and intramembrane signaling agents, and farnesylating agents (Berridge et al., 1999).
- the amounts of the hybrid proteins containing the bait and/or prey or other proteins and molecules are continuously varied or limited using exogenously activated promoters, exogenous activating agents, and other molecules including, but not limited to: (i) steroid responsive elements (SRE's), including but not limited to those sensitive to natural or synthetic estrogens (e.g., estradiol, estrone and others), androgens, progesterones, glucocorticoids (e.g., dexamethasone, cortisone, hydrocortisone and cortisol, among others), mineralocorticoids, ecdysones, metabolically inactive corticoids, other steroids (e.g., ones complementary to orphan receptors), and retinoids; and/or (ii) agonist and antagonist agents in combination with (i); (iii) any other molecules, receptors and response elements, in any or all combinations effective to provide continuously variable amounts of (iii.
- SRE's steroid responsive elements
- a hybrid protein containing the bait polypeptide (iii.b.) a hybrid protein containing the prey polypeptide, which may have been derived from a library, and/or (iii.c ) generally any molecular expression involved in genetic molecular interaction systems, such as to enable the relevant detection and analysis of the preceding biologically relevant interactions (Schena et al., 1988; Picard et al., 1990; Kralli et al, 1995; Mangelsdorf and Evans, 1995; Kliewer, 1999; Martinez et al., 1999).
- Another more preferred embodiment which forms the basis for the Examples below, comprises a novel Interaction Hybrid System (IHS) which is steroid-hormone-dependent, continuously adjustable, and contains a traditional triple reporter in a Saccharomyces cerevisiae two-hybrid system.
- IHS Interaction Hybrid System
- This relates to and novelly extends the principles and basic design of a yeast two-hybrid system as first presented and patented by Stan Fields (U.S. Patent #5,283, 173), which is incorporated by reference herein.
- Saccharomyces cerevisiae strain yHYBOO 1 was constructed containing auxotrophies for the selectable markers leu2, ade2, trpl, ura3, and arg4.
- the strain is deltaGAL4 and deltaGAL80, so as to enable the use of the GAL4 DNA-binding domain (GAL4bd) and the GAL4 transcriptional activation domain (GAL4ad) as fusions for two-hybrid interaction detection, exactly as used in the original Fields two-hybrid system.
- the strain also contains integrated human estrogen receptor and integrated rat glucocorticoid receptor genes, expressed constitutively.
- the strain is leml, which enables the use of decreased concentrations of dexamethasone in yeast, presumably by eliminating a membrane pump (Kralli et al., 1995).
- the strain contains three integrated reporters for the detection of two-hybrid interactions.
- the first is a UAS G - ⁇ cZ construct for colorimetric and quantitative assays and screening.
- the second and third are UAS G -ADE2 and UAS G -LEU2, respectively; these each enable qualitative selection for yeast that contain interacting hybrid proteins or other molecules based on rescue of nutrient auxotrophies.
- the bait in the first hybrid protein, may be fused to the carboxyl- terminal end of the GAL4bd, a DNA binding domain( Figure 2).
- This first hybrid protein may be transcribed in a continuous range of amounts over up to five orders of magnitude, and under the influence of an estrogen response element (ERE) within a minimal promoter.
- ERP estrogen response element
- This promoter- first hybrid protein construct is provided on a two-micron plasmid either under ARG4 or URA3 selection.
- the second hybrid protein may be formed by fusion of the prey protein or protein fragment, which may be derived from a library, to the carboxyl-terminal end of the GAL4ad , a transcriptional activation domain ( Figure3).
- This second hybrid protein may be transcribed in a continuous range of amounts over up to five orders of magnitude and under the influence of preferably one to six, and in the present example, three, glucocorticoid response elements (GREs) within a minimal promoter, for example, including but not limited to that from CYCl.
- GREs glucocorticoid response elements
- This promoter-second hybrid protein construct is provided on a two-micron plasmid under TRP1 selection. Both hybrid protein plasmids are also shuttle vectors containing either ampicillin or kanamycin resistance and a colEl origin of replication, which provide for manipulation in E. coli bacteria.
- Estrogen is used over a concentration range of at least about 10 "12 to 10 "8 M while dexamethasone is used over a concentration range of at least about 10 "7 to 10 "4 M.
- Estradiol has some effect on yeast growth above concentrations of 10 "6 .
- Example 1 Interaction Hybrid System adjusted to give variable quantitative reporter output without modifying the reporter system
- Table 1 shows a traditional two-hybrid system and the novel Interaction Hybrid System were done using proteins previously described in Edwards et al. (1997). Methods for analysis of the two-hybrid screen are described in Edwards et al. (1997).
- Low sensitivity assays in the IHS used 10 "10 M Estradiol and 10 "7 M Dexamethasone.
- High sensitivity assays used 10 "12 M Estradiol and 10 "4 M
- Table 1 demonstrates that different relative levels of expression of a bait and of a library
- Figure 6 is an illustration of the principle of varying reporter output in a genetic interaction system given a constant reporter set.
- Yeast colonies containing identical hybrid proteins in identical strains were observed to express reporter protein at different levels when exposed to various steroid combinations.
- Yeast cells containing SNFl and SNF4 or pelle and tube constructs, fused to the bait and prey vector (respectively, see Table 1) were plated in the corresponding minimal media in the presence of different concentrations of estradiol and/or dexamethasone.
- An integrated copy of UAS G -LacZ was used as a reporter for the interaction.
- UAS G -LacZ expression was detected by the development of blue color in yeast colonies.
- Results are those obtained uniquely in the present example and invention involving promiscuous bait and employing the improved, continuously adjustable systemcalibrated to low sensitivity in order to decrease false positives.
- Results are those comparable to ones characteristically obtained using a standard nonadjustable two-hybrid system with no difference in expression between bait and library/prey hybrid proteins.
- Table 2 shows utilizing the novel adjustable yeast interaction hybrid system introduced and described as a more preferred embodiment in the paragraphs above, a standard interaction assay on yeast medium is modified to contain various concentrations of steroid substances as shown in Table 2.
- the readout is total positives comprising the number of colonies surviving selection for interaction.
- the false positives are colonies containing proteins not interacting with the bait Pelle as determined by separate in vivo or in vitro confirmation assays, including genetic analysis and immunoprecipitation.
- Results are those comparable to ones characteristically obtained using a standard nonadjustable two-hybrid system with no difference in expression between bait and library/prey hybrid proteins.
- Results are those obtained uniquely in the present example and invention involving poor quality bait and employing the improved, continuously adjustable system calibrated to high sensitivity in order to increase total positives.
- n/a denotes "not applicable” due to no colonies surviving selection.
- Table 3 shows using the same modified interaction hybrid system as described above under Example 2, a standard interaction assay on yeast medium is modified to contain various concentrations of steroid substances. The readout is again total positives, comprising the number of colonies surviving selection for interaction. The false positives are colonies containing prey hybrid proteins not likely to interact with the bait Toll based on DNA sequence analysis.
- Example 4 - Advantages of applying the approach of Example 1 to expedite the discovery of novel interactors with the promiscuous bait, human Iraki kinase
- Example 4 demonstrates the markedly improved effectiveness and efficiency of detecting known, functionally relevant and novel interactions with promiscuous bait, including but not limited to human Iraki kinase bait.
- Two parallel sets of screens using Iraki as bait are initiated with either the Roger Brent LexA nonadjustable yeast interaction trap (Gyuris et al, 1993) and with the present adjustable interaction hybrid system.
- Using lymphocyte cDNA libraries constructed for each system 10 7 possible interactions are placed under selection. Positives are yeast colonies surviving selection and therefore containing putative proteins that would interact with Iraki. Results of the Brent system are ca.
- Example 5 Advantages of applying the approach of Example 2 to expedite the discovery of novel interactors using a poor quality bait
- Example 5 elucidates the markedly improved effectiveness of detecting novel interactions with the poor quality bait, human Fc gamma receptor 1 intracellular domain (Fc gamma Rl).
- Two parallel sets of screens using Fc gamma Rl as bait, are initiated with the Roger Brent LexA nonadjustable yeast interaction trap (Gyuris et al, 1993) in comparison with the present adjustable interaction hybrid system.
- the Roger Brent LexA nonadjustable yeast interaction trap Gauris et al, 1993
- 10 8 possible interactions are placed under selection for the standard Brent interaction system and 10 7 interactions are placed under selection for the present adjustable system.
- Positives are yeast colonies surviving selection and therefore containing putative proteins that would interact with Fc gamma Rl .
- Results of the Brent system are zero positives, allowing no possibility of further analysis.
- yeast colonies are selected as putative positives at highest sensitivity only, all of which importantly are amenable to further analysis. All 15 positives represent copies of a single putative protein interactor, which are candidates for further biochemical and genetic analysis as important modulators of B lymphocyte activation.
- Example 6 Commercial and scientific relevance
- the present adjustable system provides a markedly improved means to obtain cloned DNA sequences together with their corresponding protein structural and functional information for new and known proteins of known and novel functions that can serve as candidate drugs and drug targets. Iterative use of this system enables the improved and accelerated elucidation of entire signaling pathways linking the cell membrane to the nucleus for use in all scientifically and commercially relevant DNA-based organisms.
- the increased effectiveness and efficiency of screening for protein-protein and other relevant interactions exhibited by the present adjustable interaction hybrid system is potentially widely applicable to enable a markedly increased volume of screens per unit time and cost, as well as mass screening entailing a markedly reduced analytical load. This results in many fewer biologically irrelevant interactors, but retains and increases valuable and biologically important interactors.
- HMG-box protein by interaction with the c-Myc oncoprotein in a novel bacterial expression screen. Nucleic Acid Res. 23, 269-276. 9. Bustos, S. A.; and Schleif, R. F.; 1993. Functional domain of the AraC protein. Proc.
- Karyoplasmic interaction selection strategy A general strategy to detect protein-protein interactions in mammalian cells. Proc. Natl. Acad. Sci. 89, 7958-7962. 14. Fields, S.; and Song, O.; 1989. A novel genetic system to detect protein-protein interactions. Nature 340, 245-246.
- GAL10 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc. Natl. Acad. Sci. 79, 7410-7414. 20. Gyuris, J.; Golemis, E.; Chertkov, H; and Brent, R.; 1993. Cdil, a Human Gl and S Phase Protein Phosphatase That Associates with Cdk2. Cell 75, 791-803.
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- Molecular Biology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Bioinformatics & Computational Biology (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU10744/01A AU1074401A (en) | 1999-10-07 | 2000-10-06 | Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis |
CA002386258A CA2386258A1 (en) | 1999-10-07 | 2000-10-06 | Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis |
IL14896700A IL148967A0 (en) | 1999-10-07 | 2000-10-06 | Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis |
EP00972024A EP1222261A2 (en) | 1999-10-07 | 2000-10-06 | Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis |
JP2001528573A JP2004500805A (en) | 1999-10-07 | 2000-10-06 | Tunable sensitivity genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15807999P | 1999-10-07 | 1999-10-07 | |
US60/158,079 | 1999-10-07 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001025420A2 true WO2001025420A2 (en) | 2001-04-12 |
WO2001025420A3 WO2001025420A3 (en) | 2002-04-18 |
Family
ID=22566603
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/027677 WO2001025420A2 (en) | 1999-10-07 | 2000-10-06 | Adjustable sensitivity, genetic molecular interaction systems, including protein-protein interaction systems for detection and analysis |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1222261A2 (en) |
JP (1) | JP2004500805A (en) |
AU (1) | AU1074401A (en) |
CA (1) | CA2386258A1 (en) |
IL (1) | IL148967A0 (en) |
WO (1) | WO2001025420A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009007980A1 (en) * | 2007-07-11 | 2009-01-15 | Yeda Research And Development Co. Ltd. | Nucleic acid construct systems capable of diagnosing or treating a cell state |
US10188691B2 (en) | 2016-05-27 | 2019-01-29 | Synthex, Inc. | Protein interfaces |
US11780881B2 (en) | 2017-11-22 | 2023-10-10 | Synthex, Inc. | Compounds for selective disruption of protein-protein interactions |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997031113A1 (en) * | 1996-02-23 | 1997-08-28 | Ariad Pharmaceuticals, Inc. | Cell-based assay |
US5925523A (en) * | 1996-08-23 | 1999-07-20 | President & Fellows Of Harvard College | Intraction trap assay, reagents and uses thereof |
-
2000
- 2000-10-06 AU AU10744/01A patent/AU1074401A/en not_active Abandoned
- 2000-10-06 IL IL14896700A patent/IL148967A0/en unknown
- 2000-10-06 CA CA002386258A patent/CA2386258A1/en not_active Abandoned
- 2000-10-06 WO PCT/US2000/027677 patent/WO2001025420A2/en not_active Application Discontinuation
- 2000-10-06 JP JP2001528573A patent/JP2004500805A/en active Pending
- 2000-10-06 EP EP00972024A patent/EP1222261A2/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997031113A1 (en) * | 1996-02-23 | 1997-08-28 | Ariad Pharmaceuticals, Inc. | Cell-based assay |
US5925523A (en) * | 1996-08-23 | 1999-07-20 | President & Fellows Of Harvard College | Intraction trap assay, reagents and uses thereof |
Non-Patent Citations (1)
Title |
---|
FINLEY R L ET AL: "TWO-HYBRID ANALYSIS OF GENETIC REGULATORY NETWORKS. THE YEAST TWO-HYBRID SYSTEM" YEAST TWO-HYBRID SYSTEM ( ADVANCES IN MOLECULAR BIOLOGY ), XX, XX, 1997, pages 197-214, XP002922642 cited in the application * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009007980A1 (en) * | 2007-07-11 | 2009-01-15 | Yeda Research And Development Co. Ltd. | Nucleic acid construct systems capable of diagnosing or treating a cell state |
EP2500428A3 (en) * | 2007-07-11 | 2012-12-26 | Yeda Research and Development Co. Ltd. | Nucleic acid construct systems capable of diagnosing or treating a cell state |
US10188691B2 (en) | 2016-05-27 | 2019-01-29 | Synthex, Inc. | Protein interfaces |
US11780881B2 (en) | 2017-11-22 | 2023-10-10 | Synthex, Inc. | Compounds for selective disruption of protein-protein interactions |
Also Published As
Publication number | Publication date |
---|---|
CA2386258A1 (en) | 2001-04-12 |
JP2004500805A (en) | 2004-01-15 |
AU1074401A (en) | 2001-05-10 |
IL148967A0 (en) | 2002-11-10 |
EP1222261A2 (en) | 2002-07-17 |
WO2001025420A3 (en) | 2002-04-18 |
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