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WO2009060065A1 - Procédé de quantification d'interactions transitoires entre protéines - Google Patents

Procédé de quantification d'interactions transitoires entre protéines Download PDF

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Publication number
WO2009060065A1
WO2009060065A1 PCT/EP2008/065129 EP2008065129W WO2009060065A1 WO 2009060065 A1 WO2009060065 A1 WO 2009060065A1 EP 2008065129 W EP2008065129 W EP 2008065129W WO 2009060065 A1 WO2009060065 A1 WO 2009060065A1
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Prior art keywords
protein
substrate
binder
interaction
proteins
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Nicole Schneiderhan-Marra
Andreas Brecht
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Covalys Biosciences AG
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Covalys Biosciences AG
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • the invention relates to a method of quantifying transient interactions between proteins.
  • Interactions between different proteins are one of the major pillars of biochemical pathways. In consequence there is high interest to study such interactions in cell free, but also in cellular or even more complex systems. While certain interactions are strong and show considerable stability, even after "pulling out” such a protein complex out of an intact cell or a cell lysate, other interactions are considerably more transient and not accessible to analysis after breaking the cells. Of particular interest are interactions which are modulated by covalent modification, e.g. phosphorylation, or noncovalent modification, e.g. binding of an energy rich guanosine-triphosphate group to one of the interacting proteins, which is limited in time due to the GTPase activity of the binding protein.
  • covalent modification e.g. phosphorylation
  • noncovalent modification e.g. binding of an energy rich guanosine-triphosphate group to one of the interacting proteins, which is limited in time due to the GTPase activity of the binding protein.
  • the interaction between two proteins of interest is weak for one isoform of one of the interacting proteins (e.g. one of the proteins being non phosphorylated, or one of the proteins being bound to a guanosine diphosphate, GDP), while it is high for another isoform of one of the proteins of interest (e.g. the one being phosphorylated one or several times, or bound to an energy rich guanosine triphosphate, GTP).
  • GTP energy rich guanosine triphosphate
  • the invention relates to a method of detecting and quantifying a direct or indirect transient interaction between two different proteins, characterized in that one protein is transformed into a fusion protein with a binder protein, the other one is modified with a substrate specific for the binder protein to give a substrate protein, said fusion protein and said substrate protein are allowed to interact, the reaction product between fusion protein comprising binder protein and substrate protein comprising substrate is identified and quantified, and the result used to quantify the transient interaction between said two different proteins.
  • the invention further relates to novel fusion proteins, substrate proteins, intermediates for the preparation of substrate proteins, fusion protein - substrate protein reaction products, and assay kits for applying the method of the invention.
  • Figures 1 , 2 and 3 illustrate the assay procedures described in Examples 28, 29 and 30, respectively.
  • GDP guanosine diphosphate
  • GST glutathion-S-transferase
  • GTP guanosine triphosphate
  • PBD protein binding domain
  • S substrate (benzylguanine coupled to the antibody through a polyethylene linker)
  • SNAP binder protein (modified O 6 -alkylguanine transferase available from Covalys Biosciences)
  • * (star) spectroscopically detectable label.
  • the reference numbers 1 , 2, 3, and 4 are explained in Examples 28, 29, and 30.
  • the invention relates to a method of trapping transient interactions by converting a transient interaction into a stable interaction, which can be easily detected and quantified.
  • one potentially interacting protein is transformed into a fusion protein with a binder protein, the other one is modified with a substrate specific for the binder protein to give a substrate protein.
  • the modified proteins are allowed to interact, and any interaction is trapped by reaction of the substrate with the binder protein.
  • the reaction product of the substrate with the binder protein is detected and quantified, and the result used to quantify the transient interaction between the two potentially interacting different proteins.
  • the method described and claimed herein is selective in that it results in exclusive covalent cross-linking of two different proteins, without dimerizing one or each of the proteins, in contrast to non-selective cross-linking methods described in the prior art.
  • the method relates to a proximity dependent covalent ligation of two different proteins of interest. These two proteins may be directly involved in the interaction under study or indirectly, i.e. there may be further proteins involved in a multi-protein interaction.
  • the proximity dependent covalent ligation may, for example, happen between a protein A and a protein B, involved in a "direct" transient interaction A::B.
  • ligation may be between a protein A and a protein D out of an interaction A::B::C::D, where, for example, the interaction between B and C is transient, leading to transient proximity of all four proteins and to the proximity dependent covalent ligation of proteins A and D, depending upon the transient interaction of proteins B and C.
  • proteins A and D The interaction of proteins A and D is dependent on the (transient) interaction of protein B with protein C, and is therefore called "indirect".
  • Proteins A, B, C, and D may be all four interacting partners in the biological system studied, or three out of the four may be interacting proteins, e.g. proteins A, B, and C, with one or two further proteins, e.g. protein D (or proteins D1 and D2) being intentionally introduced for detecting the transient interaction between protein B and protein C.
  • Typical examples for such a protein D being introduced for detecting a transient interaction are antibodies, antibody fragments, but also further natural, modified or fully synthetic binder proteins known in the art. Particular examples are listed e.g. by Fiedler, M. et al.
  • Transient interactions are interactions with a half life time at or below 2 hours, preferably at or below 1 hour and most preferably at or below 30 minutes under the conditions used for the assay.
  • At least one of the potentially interacting proteins is used as a recombinant "fusion protein" between the potentially interacting protein itself and a binder unit ("binder protein"), which can lead to stable covalent binding with a substrate for the binder unit.
  • This substrate for the binder protein herewith named “binder - A - substrate”, is so chosen as to be essentially no substrate for any other protein in the system used for probing protein-protein interactions.
  • the substrate for the binder protein is then linked either directly or through a linker to the other (or another) of the potentially interacting proteins to give the "substrate protein".
  • the substrate for the binder protein is actually derived from a bifunctional substrate, and the other potentially interacting protein is first linked to a binder protein and reacted with one part of the bifunctional substrate giving a "substrate protein" wherein the other part of the bifunctional substrate is exposed as the "binder substrate”, now connected with the potentially interacting protein through a further binding protein covalently bound to one part of the bifunctional substrate.
  • this final “substrate protein” comprises the potentially interacting protein fused to a binding protein, the binding capacity of which is consumed by covalent binding to one substrate unit of a bifunctional substrate leaving the other substrate unit of the bifunctional substrate exposed for further binding, in particular for further binding to a "fusion protein” comprising a different protein.
  • Proteins are those two different proteins which undergo the “direct” or “indirect” transient interaction and, when applying the method of the invention, end up as part of the "fusion protein” and part of the “substrate protein”, respectively, in the reaction product between "fusion protein” and “substrate protein” allowing the detection and quantification of the transient interaction between said two proteins.
  • binder protein - binder substrate interactions include, but are not limited to, covalent reactions between SNAP-tag/AGT and benzylguanine derivatives (WO 02/083937) or pyrimidine derivatives (WO 2006/1 14409), CLIP-tag/AGT and benzylcytosine derivatives (WO2008/012296), Halotag and chloroalkane derivatives, and serine-beta-lactamases and beta-lactam derivatives (WO 2004/072232).
  • covalent binder protein - binder substrate interaction systems which can be used in the present invention, will depend on a synthase for forming a stable covalent bond between the binder protein and the corresponding substrate.
  • Such systems are, for example, Acyl Carrier Proteins and modifications thereof (binder proteins), which are coupled to a phosphopantheteine subunit from Coenzyme A (binder substrate) by a synthase protein (WO 2004/104588).
  • binding proteins Acyl Carrier Proteins and modifications thereof (binder proteins)
  • a synthase protein WO 2004/104588.
  • This allows to use the covalent reaction as a sensor for proximity of the binder substrate and the binder protein, and hence for proximity of "substrate protein" and "fusion protein”.
  • the system is engineered in a way that within the relevant timespan for the assay no significant covalent reaction between the binder protein and the binder substrate will occur if at least one of the two molecular species is homogeneously distributed within the sample volume.
  • the binder protein incorporated into the "fusion protein” and the protein carrying the binder substrate are in transient close proximity through a molecular interaction the likelihood of reaction between the respective binder protein and the binder substrate molecule will significantly increase.
  • the system can be adjusted to a wide range of assay conditions (assay duration, assay temperature, concentrations of reactants) by selecting an appropriate combination of a binder protein incorporated into the "fusion protein” and an appropriate binder substrate.
  • binder protein For concentrations at or below 100 nM of both binder protein and protein modified with the binder substrate (the "substrate protein"), and for an assay time at or below 30 min it would be reasonable to select a binder protein - binder substrate pair from a highly engineered and highly reactive version of AGT (such as described in WO
  • a benzylguanine substrate for concentrations above 1 ⁇ M for both of binder protein and of the protein modified with the binder substrate it will be preferable to use another combination of binder protein and of the protein modified with the binder substrate, like a pyrimidine type substrate and a lower reactivity engineered version of SNAP-tag (such as described in WO 02/083937), or a combination of an AGT variant reactive for benzylcytosines and a benzylcytosine substrate (such as described in WO2008/012296). Further options to optimize the degree of covalent interaction leading to linking the two different proteins seen in the assay are available by selecting a particular length of the linker connecting the binder substrate with one of the interacting proteins to give the "substrate protein".
  • fusion protein between one of the potentially interacting proteins and a binder unit
  • the protein or the protein part being on the most distal side of the protein-protein interaction under study is modified with a binder substrate, coupled directly or optionally through a linker or optionally through a further binding protein reacted with one part of a bifunctional substrate, to give the "substrate protein”.
  • fusion protein will be used for the recombinantly expressed fusion protein between one of the potentially interacting proteins and a binder unit (binder protein).
  • binder protein The other potentially interacting protein coupled to the corresponding binder substrate, either directly or optionally through a linker or optionally through a further binding protein reacted with one part of a bifunctional substrate, is named "substrate protein”.
  • Linker is a linking unit consisting of 1 to 300 carbon atoms, wherein up to a third of the carbon atoms may be replaced by oxygen atoms and/or nitrogen atoms and/or one or more carbon atoms may be replaced by sulfur atoms, may be linear or branched and/or comprise double bonds, triple bonds, carbocycles or heterocycles, and may carry further substituents, in particular an oxo group on a carbon atom adjacent to a nitrogen atom or an oxygen atom.
  • the linkers used are chemically stable under the conditions of the actual application.
  • the linkers do not interfere with the reaction of the substrates with the binder proteins.
  • a linker is a straight or branched chain alkylene group with 1 to 300 carbon atoms, wherein optionally (a) one or more carbon atoms are replaced by oxygen, in particular wherein every third carbon atom is replaced by oxygen, e.g. a poylethyleneoxy group with 1 to 100 ethyleneoxy units;
  • one or more carbon atoms are replaced by nitrogen carrying a hydrogen atom or further substituent, representing an amine function, or, in the case that the adjacent carbon atom is substituted by oxo, an amide function -NH-CO-;
  • one or more carbon atoms are replaced by a phenylene, a saturated or unsaturated cycloalkylene, a saturated or unsaturated bicycloalkylene, a bridging heteroaromatic or a bridging saturated or unsaturated heterocyclyl group;
  • one or more carbon atoms are replaced by a sulfur atom, representing a thioether or, if two adjacent carbon atoms are replaced by sulfur atoms, a disulfide linkage -S-S-; or a combination of two or more, especially two or three, alkylene and/or modified alkylene groups as defined under (a) to (f) hereinbefore, optionally containing substituents.
  • Substituents considered are e.g. lower alkyl, e.g. methyl, hydroxy, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, amino, lower acylamino, e.g. acetylamino or trifluoroacetylamino, halogenyl, e.g. chloro, or oxo.
  • substituents considered are e.g. those obtained when an ⁇ -amino acid, in particular a naturally occurring ⁇ -amino acid, is incorporated in the linker, wherein carbon atoms are replaced by amide functions -NH-CO- as defined under (b).
  • part of the carbon chain of the alkylene group is replaced by a group -(NH-CHR-CO) x - wherein x is between 1 and 100 and R represents a varying residue of an ⁇ -amino acid.
  • a phenylene group replacing carbon atoms as defined under (e) hereinbefore is e.g. 1 ,2-, 1 ,3-, or preferably 1 ,4-phenylene.
  • the phenylene group is further substituted by a nitro group, and, combined with other replacements as mentioned above under (a), (b), (c), (d), and (f), represents a photocleavable group, and is e.g.
  • a saturated or unsaturated cycloalkylene group replacing carbon atoms as defined under (e) hereinbefore is derived from cycloalkyl with 3 to 7 carbon atoms, preferably from cyclopentyl or cyclohexyl, and is e.g.
  • a saturated or unsaturated bicycloalkylene group replacing carbon atoms as defined under (e) hereinbefore is derived from bicyclo- alkyl with 7 or 8 carbon atoms, and is e.g.
  • bicyclo[2.2.1]heptylene or bicyclo[2.2.2]- octylene preferably 1 ,4-bicyclo[2.2.1 ]heptylene optionally unsaturated in 2-position or doubly unsaturated in 2- and 5-position, and 1 ,4-bicyclo[2.2.2]octylene optionally unsaturated in 2-position or doubly unsaturated in 2- and 5-position.
  • a bridging hetero- aromatic group replacing carbon atoms as defined under (e) hereinbefore is e.g. triazolidene, preferably 1 ,4-triazolidene, or isoxazolidene, preferably 3,5-isoxazolidene.
  • a bridging saturated or unsaturated heterocyclyl group replacing carbon atoms as defined under (e) hereinbefore is e.g. derived from an unsaturated heterocyclyl group, e.g. 3,5- isoxazolidinene, or a fully saturated heterocyclyl group with 3 to 12 atoms, 1 to 3 of which are heteroatoms selected from nitrogen, oxygen and sulfur, e.g.
  • pyrrolidinediyl piperidine- diyl, tetrahydrofuranediyl, dioxanediyl, morpholinediyl or tetrahydrothiophenediyl, preferably 2,5-dioxopyrrolidine-1 ,3-diyl (succinimido), 2,5-tetrahydrofuranediyl or 2,5- dioxanediyl.
  • a particular heterocyclyl group considered is a saccharide moiety, e.g. an ⁇ - or ⁇ -furanosyl or ⁇ - or ⁇ -pyranosyl moiety, or a succinimido group.
  • a linker is preferably a straight chain or a doubly or triply branched chain alkylene group with 6 to 25 carbon atoms optionally comprising one or more, for example 1 to 6 amide functions -NH-CO-, or a straight chain or a doubly or triply branched chain polyethylene glycol group with 3 to 100 ethyleneoxy units, optionally comprising one or more, for example 1 to 6 amide functions -NH-CO-, a urea function -NH-CO-NH-, and optionally a thioether function and a succinimido group, i.e.
  • the thioether function is preferably connected to the succinimido group.
  • a straight chain or branched linker comprising one or more polyethylene glycol groups of 3 to 12 ethylene glycol units and alkylene groups wherein carbon atoms are replaced by amide bonds, and further carrying substituted amino and hydroxy functions and/or thioether and succinimido groups.
  • Other preferred branched linkers have dendritic (treelike) structures wherein amine, carboxamide, ether and/or thioether functions replace carbon atoms of an alkylene group.
  • the length of the linker will be adjusted to the protein-protein interaction under study, and may be deduced from the molecular structures of the two different proteins.
  • the length of the linker at maximum elongation will be between 0.5 nm and 20 nm, preferably between 1 nm and 10 nm and most preferably between 2 nm and 8 nm. If desired, the linker will be so chosen such as to show considerable flexibility for interactions in a cell free test system, for example by selecting a polyoxyethylene chain linker.
  • Such a linker will also increase the solubility of the substrate combination in buffered saline solutions, preferably at least up to the mg/ml level.
  • linker - substrate combination For intracellular applications the transfer of the linker - substrate combination through the cell membrane is critical and will require the use of less flexible units, for example by selecting linkers based on saturated or unsaturated omega- aminoalkylcarboxylic acids or successive omega-aminoalkylcarboxylic acids connected through amide bonds.
  • a particularly preferred linker is a straight chain alkylene group with 6 to 25 carbon atoms comprising one or more, for example 1 to 6 amide functions -NH-CO- and optionally a urea function -NH-CO-NH- and/or double bonds and/or thioether and succinimido groups, or a straight chain polyethylene glycol group with 3 to 36 ethyleneoxy units comprising one or more, for example 1 to 6 amide functions -NH-CO- and optionally a urea function -NH-CO-NH-, and/or thioether and succinimido groups.
  • Lower alkyl is alkyl with 1 to 7, preferably from 1 to 4 C atoms, and is linear or branched; preferably, lower alkyl is butyl, such as n-butyl, sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl, ethyl or methyl. Most preferably, lower alkyl is methyl.
  • lower alkoxy the lower alkyl group is as defined hereinbefore.
  • Lower alkoxy denotes preferably n-butoxy, tert-butoxy, iso-propoxy, ethoxy, or methoxy, in particular methoxy.
  • lower acyl has the meaning of formyl or lower alkylcarbonyl wherein lower alkyl is defined as hereinbefore.
  • Lower acyloxy denotes preferably n-butyroxy, n-propionoxy, iso-propionoxy, acetoxy, or formyloxy, in particular acetoxy.
  • Lower acylamino is preferably acetylamino.
  • Halogen is fluoro, chloro, bromo or iodo, in particular chloro.
  • reaction When the reaction is carried out inside living cells, it is understood that the reaction is carried out in living cells in vitro using cells of an isolated cell lines or other cells isolated from living multicellular systems, or alternatively in vivo in animals.
  • a typical time window for incubation and potentially crosslinking between "fusion protein” and “substrate protein” for such an assay is between 5 minutes and 24 hours, more preferably between 10 minutes and 4 hours, and most preferred between 30 minutes and 2 hours. People skilled in the art are familiar with a range of assay conditions used for the study of such protein-protein interactions, including buffer compositions, and temperatures.
  • the covalent crosslinking can be stopped and preferably is stopped after a defined time by a variety of methods, e.g. either by separating the assay compounds, by changing the reaction conditions in a way that will stop further interactions (e.g. change in temperature, change in pH-value, addition of detergents, addition of denaturing agents, and similar methods well known to those skilled in the art), or by adding an agent which will compete with the interaction of the binder protein part of the "fusion protein" and its substrate.
  • a variety of methods e.g. either by separating the assay compounds, by changing the reaction conditions in a way that will stop further interactions (e.g. change in temperature, change in pH-value, addition of detergents, addition of denaturing agents, and similar methods well known to those skilled in the art), or by adding an agent which will compete with the interaction of the binder protein part of the "fusion protein" and its substrate.
  • a "fusion protein” comprising a SNAP-tag derivative and a "substrate protein” comprising a benzylguanine substrate for the interaction between a "fusion protein” comprising a SNAP-tag derivative and a "substrate protein” comprising a benzylguanine substrate, the addition of a low molecular weight benzylguanine derivative in a concentration of 10 micromolar, in particular in a concentration at least fivefold higher than the concentration of binder substrate as part of the "substrate protein” used in the assay, will stop the covalent crosslinking.
  • fusion protein and "substrate protein”
  • assay system which may be inside living cells, or in a cell lysate, or in a system consisting of purified proteins
  • significant covalent crosslinking as a result of reaction of the substrate with the binder protein will be observed within the time window selected for the assay, if the potentially interacting proteins under study are indeed interacting. That means that at least two times as much of "fusion protein” will become covalently linked to "substrate protein” than under reference conditions wherein no particular interaction between the two proteins exists.
  • Binder protein and binder substrate i.e.
  • the reactivity of the substrate for the binder protein) and the position of the substrate within the "substrate protein” are preferably so chosen as to give a more pronounced difference between the result of interacting proteins and the reference system without interaction, preferably at least three times as much, and most preferably at least five times as much.
  • the system used for detection of the transient interaction needs to be adjusted in a way giving only low crosslinking if no specific interaction takes place.
  • This is achieved by variation of the binder protein subunit (e.g. for SNAP-tag there exist variants with reactivities from above 5000 l/mol/s down to below 300 l/mol/s for benzylguanine substrates), by variation of the chemical nature of the binder substrate (e.g. SNAP-tag reactivities change by an order of magnitude from benzylguanine to substituted pyrimidines), by adjusting the concentrations of the reacting proteins, as far as this is possible in the system under study, and by adjusting the temperature and the duration of the assay.
  • the initial rate of reaction will be preferably below the 0.1% level per second.
  • the assay time window can be adapted to the interaction under study, allowing e.g. for extended interaction times if only a low percentage of interacting proteins is to be detected.
  • Other hitherto used common approaches like the non-specific crosslinking of biomolecules using aldehyde treatment do not offer this possibility.
  • Nonspecific covalent chemical binding This can be achieved by using a derivative of the binder substrate, optionally modified with a linker as defined hereinbefore, e.g. a polyethylene glycol chain, carrying a reactive group, e.g. an N-hydroxy succinimde ester function, a maleimide group or any other group reactive for proteins, and incubating this with the purified potentially interactive protein to be converted into a "substrate protein", in a ratio that will lead to an acceptable modification, preferentially in the range from one binder substrate unit per potentially interactive protein to three binder substrate units per potentially interactive protein, followed by separation of modified protein (i.e. "substrate protein") and unreacted binder substrate, in a manner well known to those skilled in the art.
  • a linker as defined hereinbefore, e.g. a polyethylene glycol chain
  • a reactive group e.g. an N-hydroxy succinimde ester function, a maleimide group or any other group reactive for proteins
  • Affinity binding The coupling of a potentially interactive protein with a binder substrate can also be achieved in a non-covalent way by using a recombinant expression of the potentially interactive protein fused to an affinity tag binding protein, and reacting such recombinantly expressed protein with the binder substrate carrying the corresponding affinity tag on the distal end.
  • a potentially interactive protein with a recombinant GST subunit, this GST subunit showing high affinity for a GSH peptide group representing an affinity target being present as part of the binder substrate, optionally linked to the binder substrate by a linker as defined hereinbefore.
  • Another example is the fusion of a potentially interactive protein with a bacterial dihydrofolate reductase, showing high affinity to a binder substrate connected with a trimethoprim group or another group representing an affinity target for dihydrofolate reductase as described in United States Patent Application 2006/021 1007, optionally attached to the binder substrate by a linker as defined hereinbefore.
  • a further example is the fusion of a potentially interactive protein with an antibody fragment, preferably with an antibody fragment with exactly one binding site for an epitope within the binder substrate, but not interfering with the ability of the binder substrate to react with its binder protein.
  • this approach can also be used for the modification of a potentially interactive protein in complex samples, provided that the binding constant of the affinity tag and the affinity target are high enough, with K D being 100 nanomolar or below. If the binder substrate is cell membrane permeable and if no intracellular competing agent is present in significant concentrations, this approach can also be used for assays inside living cells.
  • Binding through a specific, covalent substrate - protein interaction The coupling of a potentially interactive protein to the binder substrate can also be achieved in a stoichiometric way by recombinant expression of an intermediate fusion with a binder protein, this binder protein being essentially identical or being different from the binder protein used in the "fusion protein". Such an intermediate is then further reacted with one part of a bifunctional substrate providing the desired "substrate protein". A range of conditions are feasible with this approach:
  • Preloading a fusion of the potentially interactive protein with a binder unit with a homo- bivalent substrate A fusion of a potentially interactive protein with a binder unit is mixed with a significant excess of a homo-bivalent substrate. Under these conditions the coupling of one of the substrate units of a homo-bivalent substrate to every fusion of the potentially interactive protein and a binder unit will occur. The mixture is separated thereafter, e.g. on a gel filtration matrix, yielding a high yield of "substrate protein" comprising the potentially interactive protein fused to a binder unit further coupled to the binder substrate available for binding to another unit of the same binder unit type. This "substrate protein" can thereafter be used in the assay described above.
  • Preloading a fusion of a potentially interactive protein with a binder unit with a hetero- bivalent substrate A fusion of a potentially interactive protein with a binder unit is mixed with a modest excess of a hetero-bivalent substrate. The mixture is separated thereafter, e.g. on a gel filtration matrix, yielding a high yield of "substrate protein" comprising the potentially interactive protein fused to a binder unit further coupled to a substrate for a different binder unit. This "substrate protein" can thereafter be used in the assay described above.
  • a fusion of a potentially interactive protein with a binder unit with a hetero- bivalent substrate, one substrate subunit being blocked from reaction by a labile blocker in the sense of a protecting group A fusion of a potentially interactive protein with a binder unit is mixed with a modest excess of a hetero-bivalent substrate, this substrate consisting of one reactive substrate group and another substrate group being blocked by a removable chemical entity.
  • Such chemical entities can be photocleavable or photo- removable groups, groups showing reduced stability in the reducing environment inside a cell, or groups being substrates to intracellular enzymes, e.g. to esterases, which can remove the blocking group, or bio-orthogonal cleavage type reactions.
  • This approach is preferentially used inside living cells, by adding an excess of the cell permeable bivalent substrate with one reactive substrate group and the other substrate group being blocked, waiting for reaction with the fusion of the potentially interactive protein and a binder protein, and subsequently washing out the excess substrate. Thereafter the labile blocker is removed, e.g. by application of light, by use of a chemical cleaving agent, or by use of intracellular release by enzymes. Before removing the labile blocker protecting the substrate, this method allows to test an in-built reference system for the interaction between a "substrate protein" type (not displaying the substrate) and the "fusion protein".
  • fusion protein and "substrate protein”, respectively, or against further sequences comprised within the "fusion protein” or "substrate protein”, e.g. one antigenic peptide or two different antigenic peptides.
  • the protein-protein aggregates are then quantified using a sandwich fluorescent immunoassay or a similar heterogeneous phase assay.
  • a mix and measure interaction assay is possible, e.g. a FRET or a TR-FRET assay, using two different antibodies labeled with a donor compound and an acceptor compound, respectively.
  • One compound of such a detection system may be already engineered into the binder substrate as part of the assay system.
  • One example is the acceptor compound of a TR- FRET system being coupled to the binder substrate.
  • a single antibody against the "fusion protein” or against a protein tag being expressed in conjunction with the "fusion protein” and labeled with the donor compound of the TR-FRET system is sufficient.
  • a fluorescent binder substrate is used for establishing the "substrate protein” and by subsequent detection of fluorescent derivatives of "substrate protein” with increased molecular weight due to coupling of one or of several units of "fusion protein", or by using an affinity probe, e.g. a labeled antibody, against one of the proteins or optionally against a further peptide tag being present on one of the proteins.
  • an affinity probe e.g. a labeled antibody
  • detectable labels can be used, including but not limited to radioactive compounds, enzymatically active compounds, and luminescent probes.
  • this assay system can also be used to establish arrays, e.g. an array of various proteins, all showing binding to one and the same interaction partner, which depends on the metabolic situation.
  • arrays e.g. an array of various proteins, all showing binding to one and the same interaction partner, which depends on the metabolic situation.
  • a preferred way to establish such an array will be the use of bead arrays such as the xMAPTM system from Luminex Corp.
  • fusion protein and “substrate protein” may be direct, without further proteins involved, or may be more complex involving one or several further proteins.
  • the relevant transient interaction may take place between one or two proteins being different from the fusion protein and/or the binder protein.
  • target protein one further protein is considered, named "target protein”.
  • the assay can be performed in a range of different ways:
  • Target proteins may be coming from a recombinant expression system or out of cell or tissue lysates.
  • the cells or the lysates containing such "target protein(s)” may be incubated under different conditions and with different compounds added to result in various levels of affinity of the "target protein(s)” to the "fusion protein” (e.g. high affinity state or low affinity state).
  • altering the activation state of the "target protein(s)” and hence affinity to the "fusion protein” can be established during the incubation of the "target protein(s)" on the surfaces, e.g. on the preferred bead surfaces.
  • the assay is then carried out by exposing a selection of beads with particular "target proteins” on them, incubating them with a “fusion protein”, and finally detecting the amount of "fusion protein” bound to each class of beads corresponding to one kind (or two or three) of the "target proteins”.
  • Bound "fusion protein” may be detected, e.g., by further adding an antibody against “fusion protein” or against a protein tag present within the "fusion protein", this antibody being labeled or detected by a labeled secondary antibody in a way that allows detection on a suitable readout system.
  • Beads are modified with a "fusion protein” or with an antibody against “fusion protein", i.e. with an antibody against the potentially interacting protein or an antibody against the binder protein or an antibody against a protein tag present within the "fusion protein".
  • This "fusion protein” may be the same or different for a range of “target proteins”.
  • one kind or two or three kinds of activatable “target proteins” are loaded per kind of bead.
  • target proteins may be coming from a recombinant expression system or out of cell or tissue lysates.
  • the cells or the lysates containing such "target protein(s)” may be incubated under different conditions and with different compounds added to result in various levels of affinity of the "target protein(s)" to the "fusion protein” (e.g. high affinity state or low affinity state).
  • the conditions can be applied before or during binding the "target protein(s)" to the surface, e.g. to the preferred bead surface, by the "fusion protein".
  • a selection of beads with an immobilized "fusion protein” and one, two or three kinds of "target proteins” per kind of bead is used.
  • the "substrate protein” binds to the "target protein” which to a varying degree will be bound to the binder protein, allowing the proximity dependent ligation between "binder protein” and “substrate protein” to take place. Finally the amount of “substrate protein” bound to each class of beads corresponding to one, two or three kinds of “target proteins” will be detected. Bound “substrate protein” may be detected, e.g., by further adding an antibody against "substrate protein”, i.e. an antibody against the potentially interacting protein or against a protein tag present within the "substrate protein", this antibody being labeled or detected by a labeled secondary antibody in a way that allows detection on a suitable readout system.
  • an antibody against “substrate protein” i.e. an antibody against the potentially interacting protein or against a protein tag present within the "substrate protein
  • this antibody being labeled or detected by a labeled secondary antibody in a way that allows detection on a
  • One preferred application is to follow the phosphorylation of at least one kinase target, stimulating the interaction or inhibiting the interaction, but preferably stimulating the interaction between the kinase target and a further protein of interest.
  • One further preferred application is to follow the dephosphorylation of at least one phosphorylated phosphatase target, stimulating the interaction or inhibiting the interaction, but preferably stimulating the interaction between the phosphatase target and a further protein of interest.
  • RhoGTPases small GTPase
  • PBD protein binding domain
  • RhoGAPs RhoGTPase Activating Proteins
  • the invention further relates to novel fusion proteins as described hereinbefore, in particular to "fusion proteins" of potentially interacting proteins with binder proteins selected from the group consisting of SNAP-tag/AGT, CLIP-tag/AGT, Halotag, serine- beta-lactamases, and Acyl Carrier Proteins and modifications thereof.
  • binder proteins selected from the group consisting of SNAP-tag/AGT, CLIP-tag/AGT, Halotag, serine- beta-lactamases, and Acyl Carrier Proteins and modifications thereof.
  • the invention further relates to novel substrate proteins as described hereinbefore, in particular to "substrate proteins” comprising potentially interacting proteins and a substrate selected from the group consisting of benzylguanine derivatives, pyrimidine derivatives, benzylcytosine derivatives, chloroalkane derivatives, beta-lactam derivatives and Coenzyme A derivatives.
  • substrate proteins comprising potentially interacting proteins and a substrate selected from the group consisting of benzylguanine derivatives, pyrimidine derivatives, benzylcytosine derivatives, chloroalkane derivatives, beta-lactam derivatives and Coenzyme A derivatives.
  • the invention relates to such "substrate proteins” wherein the potentially interacting protein is bound to the substrate through a linker as defined hereinbefore.
  • the invention relates to "substrate proteins” wherein the potentially interacting protein is bound to a binding protein connected with one subunit of a bifunctional substrate as defined hereinbefore.
  • bifunctional substrates comprising two identical or different substrate units independently selected from pyrimidine derivatives, benzylcytosine derivatives, chloroalkane derivatives, beta-lactam derivatives, and Coenzyme A derivatives, optionally connected through a linker as defined hereinbefore.
  • Particular objects of the invention are homo-bifunctional substrates consisting of two identical pyrimidine derivatives, benzylcytosine derivatives, chloroalkane derivatives, beta-lactam derivatives, or Coenzyme A derivatives, and hetero-bifunctional substrates wherein one substrate is a benzylguanine derivative and the other substrate is selected from the group consisting of pyrimidine derivatives, benzylcytosine derivatives, chloroalkane derivatives, beta-lactam derivatives, and Coenzyme A derivatives, optionally connected through a linker, as defined hereinbefore.
  • Homo-bifunctional consisting of two benzylguanine are known.
  • the invention further relates to fusion protein - substrate protein reaction products obtained on using the method of the invention as described hereinbefore.
  • kits for the detection and quantification of transient protein interactions according to the method as described hereinbefore.
  • a kit for the detection of the interaction between a recombinant "fusion protein” comprising a binder protein subunit and a protein of interest, and a recombinant "substrate protein” comprising a different protein of interest and also comprising a binder protein subunit may, for example, contain: 1.
  • a homobifunctional substrate for crosslinking two binder units or as an alternative, the recombinant "substrate protein" being already modified with the substrate.
  • test compounds with known performance, e.g. stimulating the interaction under study or inhibiting the interaction under study.
  • FRET FRET
  • TR-FRET TR-FRET system consisting of two antibodies labeled with donor and acceptor fluorophore, respectively, and recognising part of the sequence of the recombinant "fusion protein” or part of the sequence of the recombinant "substrate protein”;
  • fusion protein a solid phase
  • a binder e.g. an antibody directed to part of the sequence of one of the interacting proteins
  • another affinity binder e.g. streptavidin
  • the homobifunctional substrate being modified with the corresponding affinity ligand, e.g. biotin
  • another affinity binder e.g. an antibody recognizing part of the sequence of the other interacting protein and carrying a label useful for detection, e.g. an enzyme, a fluorophore, a luciferase or another label known to those skilled in the art.
  • a kit for the detection of the interaction between a recombinant "fusion protein” comprising a binder protein subunit, and a "target protein”, using a further “substrate protein”, offering at least one substrate unit and having a significant affinity for the "target protein” may, for example, contain:
  • substrate protein having an affinity for the "target protein” and being covalently modified with at least one substrate for the binder protein, this substrate including optionally a linker, e.g. an antibody, this "substrate protein” potentially carrying a label for detection of complexes of the "fusion protein” and the "substrate protein”.
  • a FRET or a TR-FRET system consisting of two antibodies labeled with donor and acceptor fluorophore, respectively, and recognising part of the sequence of the recombinant "fusion protein” or part of the sequence of the recombinant "substrate protein”;
  • - or a FRET or a TR-FRET system with the acceptor label being present on the "substrate protein” and an antibody being labeled with the donor label and recognising part of the sequence of the recombinant "fusion protein”;
  • a heterogeneous phase detection system consisting of a solid phase, e.g. a microplate, modified with a binder, e.g. an antibody directed to part of the sequence of one of the interacting proteins, or another affinity binder, like e.g. streptavidin, in that case the homobifunctional substrate being modified with the corresponding affinity ligand, e.g. biotin, and another affinity binder, e.g. an antibody recognizing part of the sequence of the other interacting protein and carrying a label useful for detection, e.g. an enzyme, a fluorophore, a luciferase or another label known to those skilled in the art.
  • a binder e.g. an antibody directed to part of the sequence of one of the interacting proteins
  • another affinity binder like e.g. streptavidin
  • the homobifunctional substrate being modified with the corresponding affinity ligand, e.g. biotin
  • another affinity binder e.g. an antibody recognizing
  • a kit for the detection of the interaction between a recombinant "fusion protein” comprising a binder protein subunit, and a "target protein”, using a further “substrate protein”, this "substrate protein” offering an additional affinity unit, which is modified with a substrate comprising the ligand corresponding to the affinity unit, may contain: 1.
  • substrate protein having an affinity domain for the "target protein” and an additional affinity unit, e.g. a GST subunit, this "substrate protein” potentially being already modified with a ligand for the affinity unit, optionally extended by a linker, and a substrate for the binder protein, this "substrate protein” further potentially carrying a label for detection of complexes of the "fusion protein” and the “substrate protein", or else the substrate molecule potentially carrying a label for detection complexes of the "fusion protein” and the "substrate protein”.
  • additional affinity unit e.g. a GST subunit
  • this "substrate protein” potentially being already modified with a ligand for the affinity unit, optionally extended by a linker, and a substrate for the binder protein
  • this "substrate protein” further potentially carrying a label for detection of complexes of the "fusion protein” and the "substrate protein”
  • the substrate molecule potentially carrying a label for detection complexes of the "fusion protein”
  • agents for detection of the quantity of crosslinked protein formed in the assay such as: - A FRET or a TR-FRET system consisting of two antibodies labeled with donor and acceptor fluorophore, respectively, and recognising part of the sequence of the recombinant "fusion protein” or part of the sequence of the recombinant "substrate protein”;
  • a heterogeneous phase detection system consisting of a solid phase, e.g. a microplate, modified with a binder, e.g. an antibody directed to part of the sequence of one of the interacting proteins, or another affinity binder, like e.g. streptavidin, in that case the homobifunctional substrate being modified with the corresponding affinity ligand, e.g. biotin, and another affinity binder, e.g. an antibody recognizing part of the sequence of the other interacting protein and carrying a label useful for detection, e.g. an enzyme, a fluorophore, a luciferase or another label known to those skilled in the art.
  • a binder e.g. an antibody directed to part of the sequence of one of the interacting proteins
  • another affinity binder like e.g. streptavidin
  • the homobifunctional substrate being modified with the corresponding affinity ligand, e.g. biotin
  • another affinity binder e.g. an antibody recognizing
  • a kit for the detection of the interaction between two optionally native proteins consisting of a "fusion protein” comprising a binder protein subunit and having affinity for one of the transiently interacting different proteins, and a "substrate protein” offering a substrate for the binder protein subunit and having an affinity for another of the interacting proteins: 1.
  • An expressed recombinant "fusion protein” comprising a binder protein, this fusion protein potentially carrying a label for detection of complexes of the "fusion protein” and the "substrate protein” and having an affinity for one of the interacting proteins, e.g. a fusion protein offering a recombinant antibody fragment with affinity for one of the interacting proteins.
  • Substrate protein having an affinity domain for another of the interacting proteins and being modified with a substrate for the binder protein, this "substrate protein” further potentially carrying a label for detection of complexes of the "fusion protein” and the "substrate protein”.
  • a FRET or a TR-FRET system consisting of two antibodies labeled with donor and acceptor fluorophore, respectively, and recognising part of the sequence of the recombinant "fusion protein" or part of the sequence of the "substrate protein”;
  • - or a FRET or a TR-FRET system with the acceptor label being present on the "substrate protein” and an antibody being labeled with the donor label and recognising part of the sequence of the recombinant "fusion protein”;
  • a heterogeneous phase detection system consisting of a solid phase, e.g. a microplate, modified with a binder, e.g. an antibody directed to part of the sequence of one of the interacting proteins, or another affinity binder, such as e.g. streptavidin, in that case the substrate being additionally modified with the corresponding affinity ligand, e.g. biotin, and another affinity binder, e.g. an antibody recognizing part of the sequence of the other interacting protein and carrying a label useful for detection, e.g. an enzyme, a fluorophore, a luciferase or another label known to those skilled in the art.
  • a binder e.g. an antibody directed to part of the sequence of one of the interacting proteins
  • another affinity binder such as e.g. streptavidin
  • DIPEA diisopropylethylamine
  • GTP guanosine triphosphate
  • HA haemagglutinin
  • HPLC high pressure liquid chromatography
  • PE phycoerythrin
  • PEG polyethylene glycol
  • PEGn -(CH 2 CH 2 O) n -
  • SDS sodium dodecyl sulfate
  • Tris tris(hydroxymethyl)methylamine
  • Example 1 1 -(1 -Bromoethyl)-2-nitrobenzene (11).
  • Example 13 " 7 NPE-BG-N 3 (13) and " 9 NPE-BG-N 3 (14)
  • BG-PEG9-NHS (19) (15 mg, 0.018 mmol) and glutathione (5.5 mg, 0.018 mmol) are dissolved in 1 mL DMF with Et 3 N (2.7 ⁇ L, 0.018 mmol) and heated overnight at 31 0 C. The solvent is evaporated under vacuum and compound (20) isolated by reversed phase
  • Example 22 In vitro assay between FKBP and FRB with a benzylquanine dimer. Detection by gel shift
  • FKBP- FRB Plasmids with the genes for both proteins used for this example and subsequent examples - FKBP and FRB - are obtained from Ariad Pharmaceutical, USA.
  • the FKBP- FRB system interacts with high affinity (KD about 10 nM) in the presence of rapamycin, but shows only weak interaction in the absence of rapamycin (KD about 10 ⁇ M).
  • "Fusion protein” consists of FRB, conjugated to the gene for SNAP26, available from Covalys, followed by a single HA-tag sequence (hemagglutinin tag), followed by a nona-histidine tag (HiS) 9 .
  • Substrate protein consists of FKBP, conjugated to the gene for SNAP26, followed by a single FLAG-tag sequence, followed by a nona-histidine tag. Both proteins are expressed in E. coli and purified using Ni-NTA (nickel nitrilotriacetic acid) chromatography. A 10 ⁇ M solution of "substrate protein” is mixed with 15 ⁇ M of BG- PEG9-BG (from Example 5) and reacted for 1 h. Unreacted substrate is removed by gel permeation chromatography. Analysis by SDS chromatography shows only traces of dimerized "substrate protein".
  • Ni-NTA nickel nitrilotriacetic acid
  • fusion protein and “substrate protein” are mixed at a final concentration of 0.2 ⁇ M.
  • One sample is mixed with 0.25 ⁇ M rapamycin, while the control is incubated without rapamycin.
  • the interaction is run for 30 min, 60 min and 120 min at 37 0 C.
  • the reaction is terminated by adding 10 ⁇ M free benzylguanine.
  • Samples are separated on an SDS gel. Already in the gel the samples treated with rapamycin show much stronger crosslinking than the control sample without rapamycin, obvious from a band with increased molecular weight than the respective control samples.
  • Western blotting and detection with an anti-HA antibody the heavy band could be shown to be HA-positive, proving the presence of crosslinked "fusion protein”.
  • a semiquantitative analysis resulted in at least a fivefold increase in the protein present in the crosslinked band with rapamycin than without rapamycin.
  • Example 23 In vitro assay with CLIP-CLIP. Detection by gel shift
  • Fusion protein consists of FRB, conjugated to the gene for CLIP-tag, available from Covalys, followed by a single HA-tag sequence (hemagglutinin tag), followed by a nona- histidine tag.
  • Substrate protein consists of FKBP, conjugated to the gene for CLIP-tag, followed by a single HA-tag sequence, followed by a nona-histidine tag. Both proteins are expressed in E. coli and purified using Ni-NTA chromatography. A 10 ⁇ M solution of "substrate protein” is mixed with 15 ⁇ M BC-PEG9-BC (from Example 7) and reacted for 1 h. Unreacted substrate is removed by gel permeation chromatography.
  • Example 24 In vitro assay with SNAP-SNAP. Detection by gel shift
  • Fusion protein consists of FRB, conjugated to the gene for SNAP22, available from Covalys, followed by a single HA-tag sequence (hemagglutinin tag), followed by a nona- histidine tag.
  • Substrate protein consists of FKBP, conjugated to the gene for SNAP26, followed by a single HA-tag sequence, followed by a nona-histidine tag. Both proteins are expressed in E. coli and purified using Ni-NTA chromatography. A 10 ⁇ M solution of "substrate protein” is mixed with 15 ⁇ M CP-PEG9-CP (from Example 6) and reacted for 2 h. Unreacted substrate is removed by gel permeation chromatography.
  • Example 25 In vitro assay with antibody-benzylquanine and SNAP-construct. Detection by gel shift
  • a polyclonal rabbit-anti-FLAG antibody (A00170) from Genscript/VWR is labeled with BG- PEG9-NHS (19, Example 18) over a range of concentrations.
  • BG- PEG9-NHS (19, Example 18)
  • 2OkD By subsequent incubation with recombinant SNAP-tag about 2OkD are added per covalent modification with a BG molecule.
  • the labeling is estimated from a non reducing SDS-PAGE.
  • the modification resulting in 2 - 3 BG-derivatives per antibody is selected for further work.
  • the assay is run under the same conditions as given in Example 20. However, here a covalent crosslinking of one or several "fusion protein" molecules to the antibody is observed. Samples are separated using a non reducing SDS-PAGE.
  • a semiquantitative analysis results in at least a fivefold increase in the protein present in the crosslinked band with rapamycin than without rapamycin.
  • Example 26 Intracellular assay using CLIP, SNAP, and a BC-BG substrate with a photoprotected BG-derivative. Readout by SDS-PAGE after cell lysis.
  • HEK-293 cells are transiently cotransfected with plasmids for "fusion protein” (FKBP- SNAP-tag fusion as described in Example 20, but in a plasmid for mammalian transfection), and "substrate protein” (FRB-CLIP-tag fusion as described in Example 20, but in a plasmid for mammalian transfection).
  • FRB-CLIP fusion protein
  • the FRB-CLIP is loaded for 20 minutes with 5 ⁇ M BC-GLA- N7 NPE-BG (Example 21 ). Thereafter cells are washed carefully three times.
  • rapamycin is added to part of the cells and cells are illuminated with a 360 nm light source for 10 min for cleavage of the photoprotective group from the benzylguanine derivative. The assay is stopped after this 10 min illumination period, or after an additional incubation period in the dark of 10 min or after an additional incubation period in the dark of 30 min.
  • control cells without rapamycin and cells not illuminated, but treated for the same time with rapamycin are used. After incubation cells are lysed on ice and proteins are separated on a reducing SDS-PAGE. The difference with and without rapamycin is striking for 10 min illumination and for 10 min illumination plus 10 min chase in the dark, but was less obvious at 30 min chase indicating a significant level of background interaction happening inside the living cells.
  • Example 27 In vitro assay with antibody-benzylquanine and SNAP-construct following the increased interaction of ERK-2 and PARP-1. Detection by gel shift
  • An anti-PARP-antibody (AB3583 from Millipore) is modified with BG-PEG9-NHS from Example 18 as described in Example 25.
  • CHO cells are transfected with a fusion of ERK- 2, SNAP26, and a FLAG-tag.
  • Control cells are left unstimulated, while test cells are stimulated for 30 min with 100 ⁇ M peroxy-vanadate.
  • the cells are lysed and the anti- PARP antibody described above is added at 100 nM.
  • the lysate is incubated for 60 min at 4 0 C and subsequently, 10 ⁇ M benzylguanine is added. Readout is done by a nonreducing SDS-PAGE followed by western blot probed with an anti-FLAG antibody.
  • the amount of FLAG-tag positive high molecular weight bands (above 15OkD) is significantly increased in the cells treated with peroxy-vanadate as compared to the control cells.
  • the following three Examples (Example 28, 29 and 30) are illustrated schematically in Figures 1 , 2 and 3, respectively.
  • captured active Rho GTPase mediates proximity for linkage between BG-labeled anti-Rho GTPase antibody and SNAP fused GST-PBD.
  • the cell culture is performed in a 96-well plate.
  • Rho GTPase e.g. anti RhoA, RhoB, RhoC, Rad , Rac2
  • a less specific antibody recognizing all 3 forms of Rho (A/B/C) or Rac1/2 is coupled to carboxylated, color coded microspheres (conventional or magnetic; Luminex Inc.).
  • the coupled antibody is labeled with NHS-PEG9-BG (19 from Example 18).
  • One or several bead types are incubated for 1 h at 4 ⁇ € with cell lysates (between 5 ⁇ g and 50 ⁇ g protein per assay) that are prepared from differentially treated cells and a SNAP fused GST-tagged Rhotekin or PAK-CRIB PBD. Lysates are prepared and handled in the following buffer: 50 mM Tris-HCI, pH 7.2, 150 mM NaCI, 10 mM MgCI2, 0.1% Triton X-100, 0.5% sodium deoxycholate, 10 ⁇ g/mL leupeptin, 10 ⁇ g/mL aprotinin, 1 mM PMSF.
  • buffer 50 mM Tris-HCI, pH 7.2, 150 mM NaCI, 10 mM MgCI2, 0.1% Triton X-100, 0.5% sodium deoxycholate, 10 ⁇ g/mL leupeptin, 10 ⁇ g/mL aprotinin, 1 mM PMSF.
  • Rho GTPases are captured by the beads (No. 1 in Figure 1 ) whereas the PBDs are only bound to active (GTP-bound) Rho GTPases.
  • the Rhotekin domain is bound by Rho A, B and C and the PAK-CRIB domain is bound to Rac 1 and 2 (No. 2 in Figure 1 ).
  • PBDs do not bind to inactive (GDP-bound) Rho GTPases.
  • the GST-PBD- SNAP is subsequently linked covalently to a specific bead by the reaction with the BG- group present on the antibody (No. 3 in Figure 1 ).
  • a PE-labeled anti-GST antibody No.
  • Rho GTPase activity assay 2 As a positive control GTP ⁇ S (0.1 mM final assay concentration) is added or a constitutively active Rho GTPase is investigated in parallel. As a negative control GDP (1 mM final assay concentration) is added to the assay. Beads are read out in a Luminex analyzer. The degree of multiplexing and specificity depends on the combination of capture antibodies each coated to individual bead types and the used PBDs.
  • a SNAP fused and GST-tagged Rhotekin and PAK-CRIB PBD are immobilized on two different bead types either by an anti-GST-antibody or GSH or coupled directly to color coded microspheres (conventional or magnetic; Luminex Inc.) (No. 1 in Figure 2). Those beads are incubated for 1 h at 4 0 C with cell lysates (between 5 ⁇ g and 50 ⁇ g total protein per assay) that are prepared from differentially treated cells.
  • Lysates are prepared and handled in the following buffer: 50 mM Tris-HCI, pH 7.2, 150 mM NaCI, 1 OmM MgCI 2 , 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 ⁇ g/mL leupeptin, 10 ⁇ g/mL aprotinin, 1 mM PMSF.
  • Active Rho A, Rho B, and Rho C is captured by the Rhotekin-PBD and active Rad
  • Rac 2 and Cdc42 is captured by the PAK-CRIB PBD (No. 2 in Figure 2).
  • Inactive (GDP-bound) Rho GTPases do not bind to PBDs.
  • Anti-Rho A/B/C and anti- Rac1/2 antibodies are labeled with BG-PEG9-NHS (19) and purified by gel permeation chromatography. A mixture of both is added to the assay. The bound antibody is subsequently linked covalently to the respective bead by the reaction with the immobilized SNAP fused, GST-tagged PBD (No. 3 in Figure 2).
  • a PE-labeled secondary anti-lgG antibody (No. 4 in Figure 2) allows the detection of the covalently bound anti-Rho GTPase antibody.
  • As a positive control GTP ⁇ S (0.1 mM final assay concentration) is added or a constitutively active Rho GTPase is investigated in parallel.
  • An anti-Rho antibody is labeled with BG-PEG9-NHS (19) and purified by gel permeation chromatography.
  • SNAP-fused GST-tagged Rhotekin-PBD is expressed in E. col i and purified by affinity chromatography on GSH-Sepharose. Purified PBDs are eluted with 10 mM GSH. GSH is removed from the eluate by gel permeation chromatography.
  • the following buffer is used for the final elution of "fusion protein" from the gel permeation column: 50 mM Tris-HCI, pH 7.6, 150 mM NaCI, 10 mM MgCI2, 0.1% Triton X-100, 10 ⁇ g/mL leupeptin, 10 ⁇ g/mL aprotinin, 0.2 mM PMSF.
  • Differentially treated cells are lysed and the BG-labeled anti-Rho GTPase antibody as well as the recombinantly expressed PBD is added and incubated for 1 h at 4 0 C.
  • the buffer described in Example 29 is used for this incubation step.
  • the BG-labeled anti-Rho GTPase antibody binds to active and not active Rho GTPases.
  • Active Rho GTP bound form
  • the antibody-GST-Rhotekin-PBD complex is captured by an immobilized anti-GST antibody or GSH-coated beads (No. 3 in Figure 3).
  • a PE-labeled secondary anti-lgG antibody No.
  • This assay procedure allows the interruption of the assay before beads are added. Samples can be frozen without any loss of information. Multiplexing can be achieved by different tagging of the PBDs, capturing by respective anti tag-antibodies or counterparts.

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Abstract

L'invention porte sur un procédé consistant à détecter et à quantifier une interaction transitoire entre deux protéines différentes. Une protéine interagissant potentiellement est transformée en une protéine de fusion avec une protéine de fixation, l'autre étant modifiée avec un substrat spécifique pour la protéine de fixation pour donner une protéine de substrat. Les protéines modifiées sont amenées à interagir, et toute interaction est piégée par réaction du substrat avec la protéine de fixation. Le produit de la réaction du substrat avec la protéine de fixation est détecté et quantifié, et le résultat utilisé pour quantifier l'interaction entre les deux protéines interagissant potentiellement.
PCT/EP2008/065129 2007-11-08 2008-11-07 Procédé de quantification d'interactions transitoires entre protéines Ceased WO2009060065A1 (fr)

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US20160297820A1 (en) * 2013-09-09 2016-10-13 Agency For Science, Technology And Research Dimerizer compound
WO2016081769A1 (fr) * 2014-11-19 2016-05-26 Cold Spring Harbor Laboratory Marqueur laser: boîte à outils permettant la récupération, le contrôle et la modification spécifiques à l'espace de cellules uniques et de molécules biologiques in vivo
WO2019241361A1 (fr) * 2018-06-12 2019-12-19 Arizona Board Of Regents On Behalf Of Arizona State University Adaptation de nappa pour analyses d'imagerie par résonance plasmonique de surface
US20220042982A1 (en) * 2018-06-12 2022-02-10 Arizona Board Of Regents On Behalf Of Arizona State University Adaptation of nappa for surface plasmon resonance imaging analyses

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