WO2009049892A1

WO2009049892A1 - Polypeptides comprising an annexin core domain, compositions, methods and use thereof

Info

Publication number: WO2009049892A1
Application number: PCT/EP2008/008783
Authority: WO
Inventors: Carsten Schultz; Alen Piljic
Original assignee: European Molecular Biology Laboratory (Embl)
Priority date: 2007-10-17
Filing date: 2008-10-16
Publication date: 2009-04-23

Abstract

The present invention relates to a first polypeptide (A) comprising a recruiting polypeptide (a) comprising at least an annexin core domain or a functional variant thereof, a bait polypeptide (b) and a luminophore. The present invention also provides a composition comprising at least one first polypeptide (A) and further comprising at least a second polypeptide (B) comprising, a first target polypeptide (d) and a luminophore (e). The present invention also relates to polynucleotides, vectors, cells and methods encoding, expressing and using the first polypeptide (A) and/or said composition, respectively.

Description

POLYPEPTIDES COMPRISING AN ANNEXIN CORE DOMAIN, COMPOSITIONS, METHODS AND USE THEREOF

The present invention relates to a first polypeptide (A) comprising a recruiting polypeptide (a) comprising at least an annexin core domain or a functional variant thereof, a bait polypeptide (b) and a luminophore. The present invention also provides a composition comprising at least one first polypeptide (A) and further comprising at least a second polypeptide (B) comprising a first target polypeptide (d) and a luminophore (e). The present invention also relates to polynucleotides, vectors, cells and methods, respectively, encoding, expressing and using the first polypeptide (A) and/or said composition.

BACKGROUND OF THE INVENTION

Assays to examine the binding of proteins to one another, or assays to isolate a modulator, e.g. a compound or polypeptide, that modulates the binding of one protein to another, are useful to elucidate intracellular binding events and to screen for new binding partners of known proteins, or compounds that modulate the intermolecular binding between two or more proteins which are relevant in, e.g. a pathology. Accordingly, such assays can also be utilized in the development and identification of new therapeutic molecules. Thus, the development of techniques that measure binding between two or more molecules is extremely important.

There are several assays known in the art, which allow the assessment of protein-protein interactions. Some of these assays are carried out in vitro like, e.g. immunological assay as "pulldown" or ELISA or in vivo like, e.g. "two-hybrid" assays. In the "pull down" assay the binding of a pair of proteins of interest is determined by forming a co-precipitate with a bead linked first antibody directed against one of the binding partners and probing the precipitate - which may be run on a protein gel first - for the other binding partner with a second antibody directed against the other binding partner. The disadvantage of this technique is that the interaction occurs in vitro and often in cell extracts, wherein all protein compartimentalization is disrupted and, thus, nonspecific binding can occur. In the "two hybrid" technique a functional transcription factor is reconstituted through the interaction of two binding partners in a cell, typically yeast. The reconstitution transcription factor triggers the transcription of a selectable or detectable protein marker (e.g. Fields, S. and Song O. (1989) Nature 340:245-246). The technique has been adapted and extended to a number of situations, including examination of enzyme-substrate interactions (R. Sikorski and R. Peters (1998) Science 281:1822-1823). In general, a first fusion protein is formed of a first protein of interest and a protein comprising a DNA binding domain and a second fusion protein is formed of a second protein of interest and of a protein comprising a transcriptional activator domain. If the two proteins of interest associate, then the two transcription factors associate and

5 transcription of the detectable or selectable protein is initiated. Disadvantages of this technique are, e.g. that it requires dimerization of the first fusion protein for sequence specific DNA binding, which may inhibit interactions between the first protein of interest and the second protein of interest; that many proteins, which are recruited to the DNA behave as transcriptional activators, which leads to non-interpretable results and/or false-positives and that the interaction

0 has to occur in the nucleus, which may not be the natural environment of the protein-protein interaction to be studied.

Thus, there is a need for improved methods of measuring protein-protein interactions and methods for finding modulators of protein-protein interactions. Especially there is a need for such methods which provide a low incidence of false-positive hits, flexibility in use and the

5 ability to study multiple protein-protein interactions simultaneously and in which protein-protein interactions can be measured that occur between cytosolic and/or nuclear proteins.

SUMMARY OF THE INVENTION

Therefore, to solve above-mentioned problems and further problems associated with the !0 prior art methods, the present invention provides a first polypeptide (A) comprising, essentially consisting or consisting of (i) a recruiting polypeptide (a) comprising, essentially consisting or consisting of at least an annexin core domain or a functional variant thereof, (ii) a bait polypeptide (b); and >5 (iii) a luminophore (c).

Furthermore, the present invention provides a composition comprising at least one first polypeptide (A) according to the invention, and further comprising at least a second polypeptide (B) comprising, essentially consisting, or consisting of: (i) a first target polypeptide (d); and SO (ii) a luminophore (e).

Also provided is a polynucleotide encoding the first polypeptide (A) according to the invention, an expression vector comprising the polynucleotide encoding the first polypeptide (A) and optionally one or more polynucleotides encoding one or more second polypeptide (B), one or more further first polypeptide (C), one or more the further second polypeptide (D) and/or one

55 or more third polypeptide (E). The invention also provides a cell comprising a polynucleotide encoding the first polypeptide (A), and/or an expression vector according to the invention and optionally one or more polynucleotides encoding one or more second polypeptide (B) according to the invention, one or more further first polypeptide (C) according to the invention, one or more the further second polypeptide (D) according to the invention and/or one or more third polypeptide (E) according to the invention or expression vectors comprising these one or more polynucleotides encoding (A) and optionally (B), (C), (D) and/or (E).

Also comprised is a method of detecting a protein-protein interaction, comprising the steps: (a) providing a composition according to the invention comprising a lipid bi-layer or a cell according the invention;

(b) inducing the translocation of the first polypeptide (A) and/or of the one or more further first polypeptides (C) to a lipid bi-layer;

(c) and detecting at least one luminophore (c) and/or (e). Also provided is a method of identifying a modulator of protein-protein interaction comprising the steps of the method of the invention and further comprising the step of contacting the composition or the cell with a test compound.

Further provided is the use of the method according to the invention, for the identification of a test compound in a library of test compounds which modulates a medically relevant protein- protein interaction.

The invention provides further a method according to the invention, further comprising the step of :

(d) repeating steps (a) through (c) a plurality of times with a library that comprises a plurality of second polypeptides (B) that comprise different target polypeptides (d) and/or (f) or polynucleotides that encode such second polypeptides (B) for identifying target polypeptides (d) and/or (f) which specifically bind to the bait polypeptide (b).

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H. G. W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

A nuclear localisation signal (NLS) as used herein is a short stretch of amino acids that mediates the transport of nuclear proteins into the nucleus. Typically, an NLS signal harbours a stretch of basic amino acids. Several databases for example PROSITE and SWISS-PROT, are available to the person of skill to search for and retrieve functional NLS sequences.

In a first aspect, the invention provides a first polypeptide (A) comprising, essentially consisting or consisting of: (i) a recruiting polypeptide (a) comprising, essentially consisting or consisting of at least an annexin core domain or a functional variant thereof; (ii) a bait polypeptide (b); and (iii) a luminophore (c).

The recruiting polypeptide (a) comprises, essentially consists or consists of at least the core domain of an annexin. Annexins are a family of calcium- and phospholipid-binding proteins. Over 20 members have been found in the eukaryotic kingdom including plants as well as animals with the exception of fungi. Exemplary sequences of proteins of the annexin family are provided in public databases such as PUBMED and can be easily accessed by a skilled person. Annexins typically have molecular weights ranging between 30 and 40 kDa. At physiological cellular steady state Ca²⁺ concentrations the majority of annexin is distributed throughout the cell and is only recruited to lipid bi-layers, if the intracellular Ca²⁺ concentration is raised. The calcium- and phospholipid-binding sites that are involved in this recruitment are mostly located in the carboxy-terminal domain of the annexin. Accordingly, not the entire annexin protein is required to be included in the recruiting polypeptide (a) to recruit the first polypeptide (A) to a lipid bi-layer in response to an increase in Ca²⁺. The term "annexin core domain" refers to the minimal fragment of annexin, which is necessary and sufficient to recruit the first polypeptide to a lipid bi-layer. This ability may be tested in a number of art known methods as described, e.g. in Piljic A. and Schultz C. (2006) MCB 17:3318-3328 or as described in Example 2 below. A core domain of an annexin has at least 30%, preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, more preferably 95% or more of the activity of the respective full length annexin protein from which it is derived to recruit polypeptide (A) to a lipid bi-layer. An annexin core domain has 100% of the activity of the full length annexin, if it recruits the first polypeptide (A) at comparable or identical conditions, e.g. external Ca²⁺ concentration, same increase in intracellular Ca concentration, same amount of Ca increasing substance added, same culture conditions etc., to the lipid bi-layer to the same extent. The extent of the recruitment can be assessed by, e.g. confocal microscopy as set out in Example 3 by determining the relative amount of fluorescence distributed throughout the cell and localized to the membrane, respectively. If, for example, the fluorescence of polypeptide (A) is measured in a cell under physiological conditions along a line through the cell, the total fluorescence in that section can be determined as the area under the curve. Upon increase of the intracellular Ca²⁺ concentration polypeptide (A) and hence the fluorescence will be redistributed, i.e. peaks of increased fluorescence will appear at the lipid bi-layers of the cellular and nuclear membrane (see, e.g. Fig. 3). Thus, the extent of the recruitment to lipid bi-layers may be quantified as the percentage of the total fluorescence that is localized to the lipid bi-layers of a cell. Accordingly, when assessing the ability of an annexin core domain to recruit polypeptide (A) to the lipid bi-layer it is preferred that the percentages of the total fluorescence localized to the lipid bi-layers for a polypeptide (A) comprising the annexin core domain is compared to the percentage of the total fluorescence localized to the lipid bi-layers for a polypeptide (A) comprising the respective full length annexin. Also preferred is the measurement of the decrease in fluorescence intensity observed in the cytosol (i.e. not on the lipid bi-layer) when the polypeptide (A) is recruited to the lipid bi-layer.

As used herein, a "luminophore" can be any polypeptide or any compound, which is capable to fluoresce or to generate a detectable, preferably fluorescent signal. The term luminophore also comprises the plural, i.e. several luminophores, which are, e.g. covalently linked to each other to enhance the fluorescence signal. Herein, a luminophore is also referred to as a chromophore. A large number of auto-fluorescent polypeptides are known, which absorb and emit light at various wave-length and which can be used in the context of the present invention including, e.g. GFP, EGFP, ECFP, BFP, EYFP, CFP, dsRED, dsRED2, mCherry, and mPlum. Further preferred examples of polypeptides usable as luminophores in the context of the present invention comprise enzymes, which generate a detectable, preferably fluorescent signal,

5 e.g. photons, when brought into contact with a substrate. The result of the enzymatic reaction, thus, may be directly detectable or may lead to one or more products that can be used as a reactant that can subsequently be used to generate chemical luminescence (ECL). Examples for such enzymes are, without limitation, luciferase and horseradish peroxidase. Representative non- peptidyl luminophores include but are not limited to quantum dot nanoparticles, phycoerythrin

0 (PE), peridinin chlorophyll protein (PerCP), allophyccocyanin (APC), cyanines, oxazines, terrylene, perylene, pyrene, Alexa Fluor(R) 350, Dansyl Chloride (DNS-CI), 5- (iodoacetamida)fluoroscein (5-IAF); fluorescein 5-isothiocyanate (FITC), tetramethylrhodamine 5- and 6-isothiocyanate (TRITC)I 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 7- nitrobenzo-2-oxa-l ,3,-diazol-4-yl chloride (NBD-CI), ethidium bromide, Lucifer Yellow, 5-

5 carboxyrhodamine 6G hydrochloride, Lissamine rhodamine B sulfonyl chloride, Texas Red(TM) sulfonyl chloride, BODIPY(TM), naphthalamine sulfonic acids including but not limited to 1- anilinonaphthalene-8-sulfonic acid (ANS) and 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS), Anthroyl fatty acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid, Fluorescein- phosphatidylethanolamine, Texas red-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,

10 Fluorenyl- phosphatidylcholine, Merocyanine 540, l-(3-sulfonatopropyl)-4-[-[beta]-[2[(di-n- butylamino)-6-naphthyl]vinyl]-pyridinium betaine (Naphtyl Styryl), 3,3'- dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentyl aminostyryl)-l-methylpyridinium (di-5- ASP), Cy-3 iodo Acetamide, Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, rhodamine 800, IR-125, Thiazole Orange, Azure B, Nile Blue, Al Phthalocyanine, Oxaxine 1,4',6-

•5 diamidino-2-phenylindole (DAPI), Hoechst 33342, TOTO, acridine orange, ethidium homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin, phytofluors, Coronene, metal-ligand complexes and derivates thereof.

Each of the above components (a), (b) and (c) may be separated from each other by a

10 linker (L). The linker has the function to increase the distance between the respective components in order to allow, e.g. component (a) to be recruited to a lipid bi-layer, (b) to interact with a target protein (see below) and (c) to acquire the correct fold, required for fluorescence. It is, thus preferred that a linker is placed between each of the three components. Accordingly, in a preferred embodiment the polypeptide of the invention may have any of the following structures:

S5 (a)-L-(b)-(c), (a)-(b)-L-(c), (a)-L-(b)-L-(c), (a)-L-(c)-(b), (a)-(c)-L-(b), (a)-L(c)-L-(b), (b)-L-(a)- (C), (b)-(a)-L-(c), (b)-L-(a)-L-(c), (b)-L-(c)-(a), (b)-(c)-L-(a), (b)-L-(c)-L-(a), (c)-L-(a)-(b), (c)- (a)-L-(b), (c)-L-(a)-L-(b), (c)-L-(b)-(a), (c)-(b)-L-(a), or (c)-L-(b)-L-(a). In preferred embodiments, wherein all three components are expressed as a fusion protein, i.e. the luminophore is a polypeptide, component (c) is located between component (a) and component

5 (b), particularly preferred structures are, thus, (a)-L-(c)-(b), (a)-(c)-L-(b), (a)-L(c)-L-(b), (b)-L- (c)-(a), (b)-(c)-L-(a) or (b)-L-(c)-L-(a). In these structures in as far as polypeptides are involved the order from left to right is from N- to C-terminus. As used herein, a "linker" is preferably a polypeptide but may also refer to other chemical groups providing a spatial distance between two entities, which is sufficient to allow free rotation of the two linked entities. It is preferred that, if

0 two polypeptides or a compound and a polypeptide are connected via a linker that the linker forms covalent or non-covalent bonds, preferably covalent bonds with the polypeptide, polypeptides and/or compounds of the invention and/or that the linking of said polypeptides does not impair the function and/or conformation of at least one of the participating polypeptides and/or compounds. A linker may also comprise a polypeptide which has affinity to an annexin

5 protein. Such polypeptides are comprised in the art. Polypeptides having an affinity to, e.g. annexin V, can have an amino acid sequence of, e.g. FAKYL WEWASVR (SEQ ID NO: 18), KTCTTPAQGN (SEQ ID NO: 19) or TTPAQGN (SEQ ID NO: 20). In this case polypeptide (A) may not comprise an annexin core domain but will comprise an annexin binding linker. In this particular embodiment it is not required to introduce an annexin into the cell, since the bait

!0 polypeptide (b) and the luminophore (c) will bind to the annexin naturally comprised in the cell. In preferred embodiments, the "linker" consists of 1 to 50 amino acids, more preferably 1 to 40 amino acids, and even more preferably said polypeptide consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 amino acids. It is especially preferred that the linker comprises one or more glycine residues.

15 In a preferred embodiment of the invention, the annexin core domain of the first polypeptide (A) is from an annexin selected from the group consisting of annexin Al, A2, A4, A5 and A6 and preferably from an N- and C-terminal deletion fragment of an annexin selected from said group. Exemplary sequences of human annexins Al, A2, A4, A5 and A6 are provided herein as SEQ ID NOs: 11-15 and can be used by the skilled person to identify homologous iO annexin proteins from other species. Typically, the annexins, which are comprised by the present invention share at least 60% similarity, preferably identity to the human annexins according to SEQ ID NO: 11-15 on the best alignment, more preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or more similarity, preferably identity. Such alignment tools are well known to the person skilled in the art and can, for example, be obtained on the World Wide Web, e.g., i5 ClustalW (www.ebi.ac.uk/clustalw) or Align (http://www.ebi.ac.uk/emboss/align/index.htmn. The alignments between two sequences may be carried out using standard settings, preferably for Align EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5. Those skilled in the art understand that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment. The "best sequence alignment" between two polypeptides is defined as

5 the alignment that produces the largest number of aligned identical residues. The "region of best sequence alignment" ends and, thus, determines the metes and bounds of the length of the comparison sequence for the purpose of the determination of the similarity score, if the sequence similarity, preferably identity, between two aligned sequences drops to less than 30% over a length of 10, 20 or 30 amino acids, preferably less than 20% over a length of 10, 20 or 30 amino

0 acids, or more preferably less than 10% over a length of 10, 20 or 30 amino acids. A part of the best sequence alignment for the annexin core sequences of human Al, A2, A4, A5, and A6 is shown in Fig, 2.

In one example, annexins can bind to lipid bi-layers that comprise phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol in the p resence of calcium ions (Ca²⁺).

5 In a preferred embodiment of the invention, the annexin core domain of the first polypeptide (A) according to the invention comprises at least an amino acid sequence according to SEQ ID NOs: 1-5. More preferably the N-terminal end of the annexin core domain comprises or consists of the amino acid Phe at position 33 for annexin A2, the amino acid Phe a position 42 for annexin 1, amino acid Phe at position 16 for annexin A4, amino acid Phe at position 15 for

:0 annexin A5 and amino acid Phe at position 20 for annexin A6. More preferably the C-terminal end of the annexin core domain comprises or consists of the amino acid Asp at position 339 for annexin A2, the amino acid Asn at position 346 for annexin 1, amino acid Asp at position 321 for annexin A4, amino acid Asp at position 320 for annexin A5 and amino acid Phe at position 325 for annexin A6. More preferably the N- and C-terminus of the annexin core domain

15 comprises at least or consists of the preferred N- and C-terminal residues indicated above. In a particularly preferred embodiment it comprises at least an amino acid sequence according to SEQ ID NOs: 6-10.

In a further preferred embodiment of the first polypeptide (A) according to the invention the functional variant of an annexin core domain is: i0 (i) a polypeptide having at least 60% similarity over the entire length of an annexin core domain according to SEQ ID NOs: 1-5, (ii) a polypeptide according to (i) having N-, C- and/or internal deletions or insertions.

In either case of (i) or (ii) the resultant functional variant has at least 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, more ι5 preferably 95% or more of the lipid bi-layer recruiting activity of the respective annexin core domain according to SEQ ID NOs 1 to 5 or the larger annexin domain spanning amino acids 42 to 346 for annexin Al according to SEQ ID NO: 11, spanning amino acids 33 to 339 for annexin A2 according to SEQ IC NO: 12, spanning amino acids 16 to 321 for annexin A4 according to SEQ ID NO: 13, spanning amino acids 15 to 320 for annexin A5 according to SEQ ID NO: 14 and spanning amino acids 20 to 325 according to SEQ ID NO: 15, preferably the annexin domain according to SEQ ID NOs 6 to 10. The recruiting activity of a may be measured as set out in more detail above. Preferably the functional variant of an annexin core domain has at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity, preferably sequence identity to the annexin core domains or the large annexin fragments as set out above, when using the entire length of the annexin core domains according to SEQ ID NO: 1 to 5 or the entire length of the longer annexin fragments indicated above, preferably according to SEQ ID NO: 6 to 10. A sequence alignment that allows to determine sequence similarity and identity, respectively, is obtainable with art known tools, e.g. Align, using standard settings, preferably EMBOSS ::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5. Thus, such tools are preferably used with the amino acid sequence set forth in SEQ ID NOs: 1 to 5 or the longer annexin fragments indicated above, preferably according to SEQ ID NO: 6 to 10. In a preferred embodiment the polypeptide having the above outlined similarity, preferably identity additionally comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids at its N- and/or C-terminus. Additionally or alternatively, the polypeptide may comprise internal deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. The invention only comprises those deletion variants, which exhibit above indicated recruiting ability.

In a further preferred embodiment of the present invention the recruiting polypeptide (a) comprises, essentially consists, or consists of an annexin selected from the group consisting of annexin Al, A2, A4, A5 and A6, i.e. the full length protein. In that it is particularly preferred that the recruiting polypeptide (a) comprises, essentially consists or consists of an annexin having an amino acid sequence according to SEQ ID NOs: 11-15.

The term "bait polypeptide" refers to a amino acid chain, having a length sufficient to form a binding epitope. Typically binding epitopes consist of at least 8 consecutive amino acids. Thus, a bait polypeptide preferably is an amino acid chain with 8 or more amino acids. Its is preferred that the entire first polypeptide (A) is encodable by a nucleic acid the amino acids comprised within the bait polypeptide are encodable amino aicds. While it is possible to identify target proteins that bind to baits that comprise only one epitope, i.e. to peptides, preferably having a length between 8 to 50 amino acids, it is more preferred to use proteins or fragments thereof having a length larger than 50 amino acids. It is further preferred that the bait polypeptide (b) is selected from the group consisting of an antibody; an enzyme, e.g. a kinase, a phosphatase or a protease; a transcription factor, e.g. steroid hormone receptors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins; a cell cycle regulator, e.g. intracellular signal transduction modulators and effectors, ras-like GTPases, apoptosis- related proteins, DNA synthesis proteins, or DNA repair proteins; a receptor, e.g. neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, seven-transmembrane receptors, growth factor receptors, hormone receptors, extracellular matrix receptors; a viral protein, e.g. hepatitis C virus (HCV) proteins, HIV proteins; a lipid-binding protein; a lectin; a cytokine; a serpin; cell-surface antigens; growth factor; a heat-shock protein; a hormone; a PDZ-domain harbouring protein and a bacterial protein or binding fragments thereof. IT is one observation of the present invention that protein fragments are similarly suitable to be used as bait or target protein in the present invention.

In a preferred embodiment of the first polypeptide (A), the bait polypeptide (b) is selected from the group consisting of CaMKIIa; CaMKIIβ; Calmodulin; p53; MDM2; RELA (p65); NFKBI (plO5); NFKBIA (IKB); CDK2; CDK4; CCNAl (cyclin Al); PIK3CA (pi 1 Oa) and PIK3R1 (p85α); insulin receptor substrate 1 (IRSl); jun; fos; smad 1; smad2; smad3; smad4; ZFYVE9; CDKNlA; PCNA; HDACl; ETHE (HSCO); STAT3; PPP2RlA,reg A (PR65); PPP2R5A,reg B (B56) and PPP2CA, cat (P2CA). Further preferred is the first polypeptide (A) according to the invention, wherein the luminophore (c) is a polypeptide, a nano fluorescent particle (NFP) and/or a fluorescent dye.

In a preferred embodiment of the first polypeptide (A) according to the invention, the luminophore (c) comprises, essentially consists or consists of at least a functional fragment of a protein that is selected from the group consisting of GFP, EGFP, ECFP, BFP, EYFP, CFP, dsRED, dsRED2, mCherry, mPlum, luciferase, horseradish peroxidase and variants thereof. Preferably the luminophore (c) Such variants share a sequence similarity, preferably identity of at least 70%, preferably at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with above indicated proteins over their entire length or in the best aligned region. The sequence of these proteins is accessible from various databanks including GenBank. A functional fragment has at least 50%, 60%, 70%, 80%, 90%, 95% and preferably 100% of the fluorescence and enzymatic activity, respectively, of the full length protein from which the fragment is derived. A large number of functional fragments for above indicated polypeptides are known in the art and further can be determined by the skilled person without undue burden. In preferred embodiments of the present invention, the luminophore comprises, essentially consists, or consists of a fluorescent protein selected from the group consisting of GFP and variants thereof, especially a green fluorescent protein having a F64L, Y66H and/or S65T mutation. See also: Chalfie, et al., "Green Fluorescent Protein as a Marker for Gene

5 Expression", Science 263(5148):802-805 (1994)); enhanced GFP (EGFP; Clontech-Genbank Accession Number U55762). As mentioned, the fluorescent protein can also be blue fluorescent protein (BFP; Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; Stauber, R. H. Biotechniques 24(3):462-471 (1998); Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), and enhanced yellow fluorescent protein

0 (EYFP; Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303) or the GFP disclosed in GenBank Acces.: U62636, cyan fluorescent protein (CFP), dsRED, dsRED2, and further fluorescent proteins known as "mCherry", "mPlum", and the like. The luminescent polypeptide could also be a poly-histidine which can be rendered fluorescent by the addition of the NTA-DCF compound (where DCF is 2',7'-dichlorofluorescein). For the use of NTA-DCF

5 see e.g. Christian R. Goldsmith et. al. (2006) J Am Chem Soc. 128(2):418-419.

When the luminophore (c) is a fluorescent dye, fluorescent labelling of the polypeptides of the invention can be accomplished using a chemically reactive derivative of a fluorophore. Common reactive groups include amine reactive isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine reactive succinimidyl esters such as

•0 NHS -fluorescein, and sulfhydryl reactive maleimide activated fluors such as fluorescein-5- maleimide. Reaction of any of these reactive dyes with another molecule results in a stable covalent bond formed between a fluorophore and a labelled molecule. Reactive fluorescent dyes are available from many sources. They can be obtained with different reactive groups for attachment to various functional groups within the target molecule. They are also available in

!5 labelling kits that contain all the components to carry out a labelling reaction. Common fluorescent dyes comprise fluorescein, rhodamine, Alexa fluors and Dylight fluors.

Depending on the recruiting polypeptide (a), bait polypeptide (b) and luminophore (c) used in the invention it may be advantageous to reduce steric hindrance and optimize binding and recruitment by altering the order in which (a), (b) and (c) are linked to each other in the

!0 polypeptide (A). Thus, in a further preferred embodiment of the first polypeptide (A) the luminophore (c) is a polypeptide and the polypeptides (a) (b) and (c) in said first polypeptide (A) are fused to each other, optionally via one or two linkers, from N- to C-terminus in the order (a)- (b)-(c), (a)-(c)-(b), (b)-(a)-(c), (b)-(c)-(a), (c)-(a)-(b) or (c)-(b)-(a). The most preferred first polypeptides (A) have from N- to C-terminus the following structure: (b)-(c)-(a), (a)-(c)-(b), (b)-

15 Ll-(c)-(a), (a)-(c)-Ll-(b), (b)-Ll-(c)-L2-(a), or (a)-Ll-(c)-L2-(b). Linker Ll preferably has a length of between 1 to 10 amino acids and linker L2 preferably has a length of 1 to 10 amino acids.

An advantage of the first polypeptide (A) of the present invention is that it can be recruited also to nuclear membranes when the Ca²⁺ ion concentration in the nucleus increases

5 over physiological steady state concentrations that are normally present in the cell nucleus. As a consequence, the first polypeptide (A) can be used in protein-protein binding assays in vivo without requiring that the first polypeptide (A) must comprise a nuclear localization signal (NLS) sequence. This is especially useful when, e.g. the bait polypeptide (b) is a transcription factor. Therefore, in a preferred embodiment of the invention, the first polypeptide (A) of the

0 invention does not comprise a nuclear localization signal (NLS) sequence.

In a related aspect, the invention provides a composition comprising at least one first polypeptide (A) according to the invention, and further comprising at least a second polypeptide (B) comprising, essentially consisting, or consisting of: (i) a first target polypeptide (d); and

.5 (ii) a luminophore (e).

When using the composition of the invention in an assay which studies homotypic interactions such as e.g. the homo-dimerization of receptors or receptor fragments, it is advantageous when the first target polypeptide (d) is identical to the bait polypeptide (b). Thus, preferred is the composition of the invention, wherein the first target polypeptide (d) is identical

JO or different to the bait polypeptide (b).

Depending on the first target polypeptide (d) and luminophore (e) used in the invention it may be advantageous to reduce steric hindrance and optimize binding and recruitment by altering the order in which (d) and (e) are linked to each other in the polypeptide (B). In a further preferred embodiment of the second polypeptide (B) the luminophore (e) is a polypeptide and

J5 the polypeptides (d) and (e) in said second polypeptide (B) are fused to each other, optionally via a linker, from N- to C-terminus in the order (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d).

The luminophore can be any of the luminophores taught above, however, in a preferred embodiment of the second polypeptide (B) according to the invention, the luminophore (e) comprises, essentially consists or consists of at least a functional fragment of a protein that is

50 selected from the group consisting of GFP, EGFP, ECFP, BFP, EYFP, CFP, dsRED, dsRED2, mCherry, mPlum, luciferase, horseradish peroxidase and variants thereof. Such variants share a sequence similarity, preferably identity of at least 70%, preferably at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with above indicated proteins over their entire length or in the best

55 aligned region. The sequence of these proteins is accessible from various databanks including GenBank. A functional fragment has at least 50%, 60%, 70%, 80%, 90%, 95% and preferably 100% of the fluorescence and enzymatic activity, respectively, of the full length protein from which the fragment is derived. A large number of functional fragments for above indicated polypeptides are known in the art and further can be determined by the skilled person without

5 undue burden.

In preferred embodiments of the present invention, the luminophore (e) comprises, essentially consists or consists of a fluorescent protein selected from the group consisting of GFP and variants thereof, especially a green fluorescent protein having a F64L, Y66H and/or S65T mutation. See also: Chalfie, et al., "Green Fluorescent Protein as a Marker for Gene Expression",

0 Science 263(5148):802-805 (1994)); enhanced GFP (EGFP; Clontech-Genbank Accession Number U55762). As mentioned, the fluorescent protein can also be blue fluorescent protein (BFP; Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; Stauber, R. H. Biotechniques 24(3):462-471 (1998); Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), and enhanced yellow fluorescent protein (EYFP;

5 Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303) or the GFP disclosed in GenBank Acces.: U62636, cyan fluorescent protein (CFP), dsRED, dsRED2, and further fluorescent proteins known as "mCherry", "mPlum", and the like. The luminescent polypeptide could also be a poly-histidine which can be rendered fluorescent by the addition of the NTA-DCF compound (where DCF is 2',7'-dichlorofluorescein). For the use of NTA-DCF

:0 see e.g. Christian R. Goldsmith et. al. (2006) J Am Chem Soc. 128(2):418^U9.

When the luminophore (e) is a fluorescent dye, fluorescent labelling of the polypeptides of the invention can be accomplished using a chemically reactive derivative of a fluorophore. Common reactive groups include amine reactive isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine reactive succinimidyl esters such as

'.5 NHS-fluorescein, and sulfhydryl reactive maleimide activated fluors such as fluorescein-5- maleimide. Reaction of any of these reactive dyes with another molecule results in a stable covalent bond formed between a fluorophore and a labelled molecule. Reactive fluorescent dyes are available from many sources. They can be obtained with different reactive groups for attachment to various functional groups within the target molecule. They are also available in i0 labelling kits that contain all the components to carry out a labelling reaction. Common fluorescent dyes comprise fluorescein, rhodamine, Alexa fluors and Dylight fluors.

Depending on the site of interaction between the bait polypeptide (b) and the first target polypeptide (d) it is possible that the recruiting polypeptide (a) or luminophore (c) of polypeptide A and/or the luminophore (e) of polypeptide (B) interfere with the binding sites of the bait

15 polypeptide (b) and the first target polypeptide (d). To account for potential steric hindrance it is preferred that various combinations of polypeptide (A) and polypeptide (B) are tested in interaction assays. Thus in preferred embodiments of the composition of the present invention: (i) luminophore (c) is N-Terminal and the bait polypeptide is C-terminal within polypeptide (A) and the luminophore (e) is N-terminal and the first target polypeptide (d) is C-terminal, (ii)

5 luminophore (c) is N-Terminal and the bait polypeptide is C-terminal within polypeptide (A) and the luminophore (e) is C-terminal and the first target polypeptide (d) is N-terminal, (iii) luminophore (c) is C-Terminal and the bait polypeptide is N-terminal within polypeptide (A) and the luminophore (e) is N-terminal and the first target polypeptide (d) is C-terminal, and (iv) luminophore (c) is C-Terminal and the bait polypeptide is N-terminal within polypeptide (A) and

0 the luminophore (e) is C-terminal and the first target polypeptide (d) is N-terminal. Particularly preferred combinations are as follows: (a)-L-(b)-(c), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (a)-(b)-L-(c), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (a)-L-(b)-L-(c) and (d)-(e), (e)-(d), (d)-L- (e) or (e)-L-(d); (a)-L-(c)-(b), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (a)-(c)-L-(b), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (a)-L(c)-L-(b), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (b)-L-(a)-

5 (c), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (b)-(a)-L-(c), and (d)-(e), (e)-(d), (d)-L-(e) or (e)- L-(d); (b)-L-(a)-L-(c), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (b)-L-(c)-(a), and (d)-(e), (e)- (d), (d)-L-(e) or (e)-L-(d); (b)-(c)-L-(a), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (b)-L-(c)-L- (a), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (c)-L-(a)-(b), and (d)-(e), (e)-(d), (d)-L-(e) or (e)- L-(d); (c)-(a)-L-(b), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (c)-L-(a)-L-(b), and (d)-(e), (e)-

10 (d), (d)-L-(e) or (e)-L-(d); (c)-L-(b)-(a), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d); (c)-(b)-L-(a), and (d)-(e), (e)-(d), (d)-L-(e) or (e)-L-(d);or (c)-L-(b)-L-(a) and (d)-(e), (e)-(d), (d)-L-(e) or (e)- L-(d). Additionally, it is possible to test an inversion of the orientation, i.e. that bait polypeptide (b) becomes the first target polypeptide (d) and conversely that the first target polypeptide becomes the bait polypeptide. In some cases this inverse orientation may allow an interaction to

15 occur that would otherwise not be possible.

The composition according to the invention can also be used to measure protein-protein interactions within and/or between entire multiprotein complexes. To that end it is preferred several different luminophores can be used with each polypeptide to allow the setup of a multiparameter assay. Thus, the luminophores used can all be identical molecules or a

(0 multiplicity of different molecules having different light absorption and/or fluorescence characteristics. Accordingly, in a preferred embodiment the composition of the invention further comprises one or more further first polypeptides (C). In this embodiment it is possible to test the, e.g. ability of the further first polypeptide (C) to compete with the first polypeptide (A) for binding to a second polypeptide (B) or to test the binding of polypeptide (A) to (B) and at the

S 5 same time the binding of polypeptide (C) to (D). Accordingly, it is preferred that the composition of the invention further comprises one or more further second polypeptides (D), i.e. each comprising, essentially consisting or consisting of a target polypeptide and a luminophore. Alternatively two, three, four, five, six, seven or eight different second polypeptides each comprising a different target polypeptide are bound to the bait polypeptide in the first polypeptide (A). Such compositions enable a skilled person to mix and match different luminophores, bait polypeptides (b) and/or target polypeptides (d) to measure multiple polypeptide-polypeptide interactions simultaneously in one assay. Thus, it is particularly preferred that the composition comprises one first polypeptide (A) and additionally comprises two, three, four, five, six, seven, eight, nine, ten or more second polypeptides (B, D, etc.), v.'herein each polypeptide (A, B, D etc.) preferably comprises a different luminophore. It is apparent to the skilled person that the respective bait polypeptide and target proteins can change their position, i.e. a target protein may become a bait protein or vice versa.

Protein-protein interactions can also be indirect, e.g. by involving a third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth second polypeptide such that itself is not labeled with a luminophor, for example the bait polypeptide binds a third, fourth, fifth, sixth, seventh, eighth, ninth and/or tenth protein, which alone or together bind to the second polypeptide (B). When in such case the bait polypeptide has no binding affinity to the target polypeptide, a third and preferably further protein(s) may be provided in trans or may be already present in the cell. Therefore, in a preferred embodiment of the composition of the invention, the composition ' further comprises one or more third polypeptides (E) comprising, essentially consisting or consisting of a target protein (f) without luminophore.

In preferred embodiments of the present invention, the luminophore (e) of the Composition of the invention is a polypeptid, a nano fluorescent particle (NFP) and/or a fluorescent dye. Preferably the luminophore is as set out in more detail above for luminophore (c')- It is preferred that both luminophores (c) and (e) are different and, thus, can be simultaneously assessed in one assay.

Also preferred is the composition of the present invention, wherein at least two of the

/lun..nophores are capable of fluorescence resonance energy transfer (FRET). The term "FRET" refers to an energy transfer mechanism between two chromophores. During said energy transfer, a donor chromophore in its excited state can transfer energy by a non-radiative, long-range dipole-dipole coupling mechanism to an acceptor chromophore, if it is localized in close proximity to the donor (typically <10 nm). The FRET efficiency (E) is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event:

where kεr is the rate of energy transfer, k/thε radiative decay rate and the k_t are the rate constants of any other de-excitation pathway. FRET technology is well known in the art and detailed information can be e.g. obtained in: Joseph R. Lakowicz, "Principles of Fluorescence

5 Spectroscopy", Plenum Publishing Corporation, 2nd edition (July 1, 1999).

In a more preferred embodiment, at least two luminophores which are capable of fluorescence resonance energy transfer (FRET) are covalently bound to the bait polypeptide (b), to the one or more target protein (d) and/or to the one or more target protein (f). This provides a first polypeptide (A) or a composition of the invention which allows measuring of not only

0 protein-protein interaction but also of additional changes that may occur in the conformational state of the bait polypeptide (b), the one or more target protein (d) and/or to the one or more target protein (f) in response to the binding. Additionally, the binding of the first polypeptide (A) to the second polypeptide (B) may bring two chromophores together, e.g. on the bait polypeptide and on the target polypeptide, in such that FRET occurs. In this case it is possible to quantify the

5 percentage of first polypeptides (A) that are bound to the lipid bi-layer and that are bound to the second polypeptide (B), e.g. the total fluorescence of the first polypeptide will be localized to the lipid bi-layers in the cells but only a certain percentage will show a shift of fluorescence emission due to FRET after binding.

In a further preferred embodiment, the bait polypeptide (b) of the composition according

!0 to the invention is capable of specifically binding to the one or more target protein (d) and/or (f). Specific binding between two or more polypeptides can be measured by methods described herein but also by alternative means such as two-hybrid assays, pull-down assays and the like. Preferably, the dissociation constant K^ between the bait and at least one target polypeptide is smaller than 1000 nM, 100 nM, 10 nM or smaller than 1 nM, More preferably the dissociation

!5 constant K^ between the bait and at least one target polypeptide is smaller than 1000 μM, 100 μM or smaller than 10 μM.

In a preferred embodiment, the composition according to the invention further comprises a lipid bi-layer. Preferably the lipid bi-layer is a plasma-membrane of a cell, the nuclear- membrane of a cell the membrane of the endoplasmic reticulum of a cell, a liposome and/or a

!0 lipid membrane on a solid support. It is well known in the art how to produce liposomes and/or preferably lipid membranes on a solid support that comprise a lipid bi-layer, which may be used in the context of the present invention.

In a preferred embodiment of the composition of the invention, the target protein (d) and/or (f) is each individually selected from the group consisting of CaMKIIa; CaMKIIβ; Calmodulin; p53; MDM2; RELA (p65); NFKBI (plO5); NFKBIA (IKB); CDK2; CDK4; CCNAl (cyclin Al); PIK3CA (pl lOα) and PIK3R1 (p85α); insulin receptor substrate 1 (IRSl); jun; fos; smad 1; smad2; smad3; smad4; ZFYVE9; CDKNlA; PCNA; HDACl; ETHE (HSCO); STAT3; PPP2R1 A,reg A (PR65); PPP2R5A,reg B (B56) and PPP2CA, cat (P2CA). A further aspect of the invention is a polynucleotide encoding the first polypeptide (A) according to the invention. The invention further comprises polynucleotides, which hybridize to the complementary strand of the polynucleotides encoding the first polypeptide (A) under stringent conditions.

"Hybridizing under stringent conditions" means that a nucleic acid is capable of hybridizing under conditions as described in T. Maniatis et al. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory (1989) or the like. For example, it refers to the capability of hybridization under the following conditions. A membrane onto which a nucleic acid is immobilized is incubated with a probe in 6 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400 and 0.01% denatured salmon sperm nucleic acid at 50 DEG C for 12 to 20 hours. After incubation, the membrane is washed in 2 x SSC containing 0.5% SDS at 37° C while changing the SSC concentration down to 0.1 x and the temperature up to 50° C until the signal from the immobilized nucleic acid can be distinguished from background, and the probe is then detected. The activity of the protein encoded by the thus obtained novel nucleic acid is determined as described above and below, thereby confirming whether or not the nucleic acid is the nucleic acid of interest.

If a codon used in a nucleic acid encoding a polypeptide of the present invention is one which is rarely or not used by the translation machinery in the host-cell or non human organism, the expression level of the polypeptide may be low. In this case, the codon may be artificially converted into one which is frequently used in the host cell or non-human organism without altering the encoded amino acid sequence to improve the expression level of the polypeptide.

Also provided is an expression vector comprising the polynucleotide of the invention and optionally one or more polynucleotides encoding one or more second polypeptide (B), one or more further first polypeptide (C), one or more of the further second polypeptide (D) and/or one or more third polypeptide (E) according to the invention. The expression of several different genes from one promoter may be achieved by separating two or more polynucleotides to be expressed by one or more internal ribosomal entry sites (IRES). It is preferred that a polypeptide (A) and a polypeptide (B), which are assayed for their binding to each other are expressed at a comparable level. It is, thus, preferred, that two or more polynucleotides encoding the different interaction partners are placed under the common control of one promoter and are separated by an IRES. In a preferred embodiment the polynucleotide comprises a polynucleotide encoding polypeptide (A) and (B); (A), (B) and (C); or (A), (B), (C) and (D).

The term "vector" as used herein includes any vector known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral, retroviral, adenovirus-associated-virus (AAV) or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. The term "expression vector" refers to a vector that contains a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence and is capable of inducing protein expression in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Expression vectors for use in mammalian cells preferably include a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and/or transcriptional terminator sequences. To obtain stable expression for an extended period of time the expression vector may either be capable of integrating into the host genome or it may contain an origin of replication (ORI). ORIs suitable for stable maintenance of expression vectors are well known in the art and comprise, e.g. for yeast cells CEN and ARS and for mammalian cells ORIs derived from extra-chromosomally replicating viruses, such as Simian Virus 40 (SV40), Polyoma, Adeno, Cytomegalic virus (CMV), vesicular stomatits virus (VSV), or bovine papilloma virs (BPV).

It is well known in the art how to direct expression in various cell systems by choosing a promoter appropriate for the respective host cell system. For mammalian expression, the promoters may be derived from the genome of mammalian cells (e. g., metallothionein promoter or GAPDH) or from mammalian viruses (e.g., the adenovirus late promoter; CMV Immediate Early promoter, SV40 promoter, or the vaccinia virus 7.5K promoter). Further, it is also possible, and may be desirable, to utilize promoter and/or control sequences normally associated with the bait and/or the target protein(s) respectively used in the fusion proteins of the present invention provided such control sequences are compatible with the host cell system used.

A number of viral based promoters may be utilized, for example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently from CMV and SV40. The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hindlϊl site toward the BgHl site located in the viral origin of replication.

Specific initiation signals may also be required for efficient translation of the coding polynucleotide sequences encoding polypeptides of the present invention. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements and transcription terminators. In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site (e.g., 5'- AATAAA-3¹) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.

For long-term, high-yield expression of a polypeptide of the invention, a stable expression is preferred. For example, cell lines that stably express constructs encoding a polypeptide of the present invention may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transiently transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA comprising the vector, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. A number of selection systems may be used including, but not limited to, the herpes simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase (aprt) genes, in tk-. hgprt-or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neomycin (neo), that confers resistance to the aminoglycoside G-418; and hygromycin (hygro), that confers resistance to hygromycin.

Also provided is a cell comprising a polynucleotide of the invention, and/or an expression vector according to the invention and optionally one or more polynucleotides encoding one or more second polypeptide (B), one or more further first polypeptide (C), one or more further second polypeptide (D) and/or one or more third polypeptide (E) or expression vectors comprising these one or more polynucleotides. Cells usable in the present invention may be any type of cell, however, the cell is preferably an eukaryotic cell, e.g. animal, yeast, or a plant cell, which comprises a lipid bi-layer. For example, in some embodiments, the cell may be a stem cell, preferably not a human embryonic stem cell, a primary skin fibroblast, a granulosa cell, a primary fetal fibroblast, a stem cell, a germ cell, a somatic cell, a fibroblast or a non-

5 transformed cell from any desired organ or tissue. In some embodiments, the cell may be a cell from which a plant may be generated, such as for example, a protoplast. The cell may be an isolated cell, e.g. an established cell line or primary cell, or may be in its natural environment, e.g. a tissue explant or in an animal. Preferred cell lines include but are not limited to those of fibroblast origin, e. g. BHK, CHO, BALB, or of endothelial origin, e. g. HUVEC, BAE (bovine

0 artery endothelial), CPAE (cow pulmonary artery endothelial), HLMVEC (human lung microvascular endothelial cells) or of pancreatic origin, e. g. RIN, INS-I, MIN6, bTC3, aTC6, bTC6, HIT, or of hematopoietic origin, e. g. primary isolated human monocytes, macrophages, neutrophils, basophils, eosinophils and lyphocyte populations, AML- 193, HL-60, RBL-I, adipocyte origin, e. g. 3T3-L1, neuronal/neuroendocrine origin, e. g. AtT20, PC12, GH3, muscle

5 origin, e. g. SKMC, AlO, C2C12, renal origin, e. g. HEK 293 or LLC-PKl . In a further preferred embodiment the cell of the invention is part of the living tissue of an explant or in a non-human organism. When the cell is in a non-human organism, it is to be understood that the methods of the invention are not to be used as a diagnostic or for the therapy of said organism. For example, transgenic nematodes expressing the polypeptides of the invention comprising, e.g., human bait

10 and human target polypeptides, can be contacted with test compounds to identify a test compound which modulates the bait-target polypeptide interaction. Such identified test compound will be therapeutic for humans but not for nematodes such as C. elegans.

In a further aspect, the invention provides a method of detecting a protein-protein interaction, comprising the steps:

Ϊ5 (a) providing a composition according to the invention or a cell according to the invention;

(c) and detecting at least one luminophore (c) and/or (e).

It is preferred that the lipid bi-layer in the method according to the invention is (0 permeabilized before, during or after step (b). A permeabilization of the lipid bi-layer of a cell, for example, permits charged compounds which are not able to cross the intact plasma membrane of a cell entry into the interior of the cell.

It is furthermore preferred that step (b) of the method according to the invention comprises the contacting of the composition or the cell with a solution comprising a substance

15 that increases the Ca²⁺ ion concentration in comparison with the Ca²⁺ ion concentration in the composition or the cell in step (a). Preferably, the substance is selected from the group consisting of Ca²⁺, in particular CaCl₂₎ calcium lactate, calcium gluconate, calcium malate, ionomycin, calcium ionophore A23187, calcium ionophore 8-Br-A23187 and thapsigarin. While free Ca²⁺ ions can only enter a cell if the plasma membrane is permeabilized, the calcium ionophores can 5 also be used on intact cells. For example, ionomycin is a calcium ionophore that comes from Streptomyces conglobatus. It is used to raise the intracellular level of Ca ⁺. Ionomycin is an ionophore that selectively shuttles Ca²⁺ ions across lipid bi-layers. In addition to equilibrating Ca²⁺ across the plasma membrane, ionomycin molecules insert themselves into the membranes of intracellular compartments, causing an equilibration of their Ca²⁺ with the cytosol. As 0 illustrated in figure 3 below, an increase in intracellular calcium ions translocates the recruiting polypeptide of the invention to lipid bi-layers such as the plasma membrane and/or internal lipid membranes.

In a preferred embodiment, step (c) of the method according to the invention is carried out before, during and/or after step (b). Thus, the detecting of a luminophore can also be carried

5 out several times, for example before and after inducing the translocation of the first polypeptide

(A). The fluorescence signals obtained by a measurement / detection before step (b) are useful as a reference. When the bait polypeptide is not capable of binding the one or more target polypeptides, it is thus expected that only luminophore (c) translocates to the lipid bi-layer while the luminophores of the one or more target polypeptides remain in the cytosol or in the assay

10 solution (if the assay is carried out in a cell-free system).

Further preferred is the method according to the invention, wherein the at least one luminophore is detected by measuring the fluorescence emission. If at least two luminophores are capable of fluorescence resonance energy transfer (FRET), one can also detect changes in the efficiency of fluorescence resonance energy transfer (FRET), for example, upon inducing the Ϊ5 translocation in step (b) and/or after contacting the composition or the cell with a test compound (see below). Thus, the skilled person obtains data which reveal if one or more of the polypeptides used in the method according to the invention has undergone a conformational shift.

In a further preferred embodiment of the method of the invention in step (c) at least one »0 luminophore is detected, which is bound to the lipid bi-layer.

In a further preferred embodiment of the method of the invention in step (c) at least one luminophore is detected, which is not bound to the lipid bi-layer. For example, if, upon carrying out step (b), a decrease in the emission intensity of a luminophore bound to one or more target polypeptides is detected in the cytosol (or the assay buffer in an in vitro assay), this is an indication that the target protein has translocated together with the bait polypeptide to the lipid bi-layer.

In a further preferred embodiment the detection is carried out by utilizing a device selected from the group consisting of a fluorescent plate reader, a fluorescence activated cell

5 sorting (FACS) apparatus, a photomultiplier tube, a linear diode array, a video camera and a charge-coupled device (CCD).

In a preferred aspect the method of the invention is used to determine the relative contribution of proteins of a protein complex to the interaction within the protein complex, i.e. the interactions among the proteins in the complex. To that end it is preferred that each protein

0 known to be or suspected to be comprised in a protein complex is expressed as bait and target protein, respectively, wherein in each case it is preferred that the bait protein is N-terminally and C-terminally fused, respectively, to the other components comprised in the first polypeptide (A) and second polypeptide (B), respectively. Accordingly, if the interaction of a given protein X, with a second protein Y is tested, it is preferred that the following combinations are tested

5 (linkers may be present between each element as outlined above): (i) recruiting polypeptide (a)- luminophore (c)-X with Y-luminophore (e), (ii) recruiting polypeptide (a)-luminophore (c)-X with luminophore (e)-Y, (iii) X-luminophore (c)-recruiting polypeptide (a) with Y-luminophore (e), and (iv) X-luminophore (c)-recruiting polypeptide (a) with luminophore (e)-Y. If a direct or indirect interaction between X and Y occurs, translocation will be detectable in step (c) of the

>0 method of the invention. If the respective contribution of a third protein "Z" is to be determined a further experiment can be conducted, wherein the interaction of Z with X and Y, respectively, is tested using in each case at least the four above indicated particularly preferred combinations. At the end of such an assessment it can be concluded whether X interacts with Z and/or Y individually and whether Y interacts with Z individually. In some cases no interaction between

55 X, Y and Z may be detected or only between two of the components. In this case it is envisioned in a preferred embodiment of the method of the invention that one protein of the potential interacting proteins is expressed as first polypeptide (A) and the other two proteins are expressed either both as second polypeptides (B) or one is expressed as second polypeptide (B) and one as third polypeptide (E). This approach may detect an interaction between, e.g. protein X and

10 protein Y that only occurs in the presence of protein Z. This may be the case since only the interaction of X and Z provides a binding scaffold for protein Y. If the protein complex to be analyzed comprises more than three protein components, the method of the present invention allows testing each individual interaction of the components of the protein complex and each combination of the components of a protein complex. Thus, preferably the method of the present

S 5 invention is used to determine the interaction, in particular the hierarchy of the interaction of the components, preferably the proteins, within a protein complex. It is noted that the term "protein complex" preferably refers to complex formed entirely from proteins but may also comprise complex that predominantly comprise proteins but may also, e.g. comprise lipids or sugars.

Another aspect of the present invention relates to a method of identifying a modulator of

5 protein-protein interaction comprising the steps of the method of the invention as described above, and further comprising the step of contacting the composition or the cell with a test compound. The test compound is a modulator, if it decreases or increases the binding affinity between the one or more bait polypeptides and the one or more target polypeptides of the invention. Such increase or decrease may be due by sterical hindrance or alternatively by

0 allosteric modulation of the respective binding pockets on the bait and the target polypeptides.

Preferably, the contacting occurs prior, concomitantly or after steps (a), (b) or (c). Thus, in one example, the composition or cell is first contacted with the test compound and then, at a later point in time, preferably after 10, 20, 30, 40, 50 or 60 seconds and more preferably after 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes, step (b) is carried out. In another example, step (b) is carried out

5 before the compound or cell is contacted with the test compound. In this case, an inhibitor of binding will release the second polypeptide (B) due to interference with the interaction between target polypeptide (d) and bait polypeptide (b) at the lipid bi-layer and it will diffuse back into the cytosol or into the assay medium and away from the lipid bi-layer.

In a further preferred embodiment of the method according to the invention the

!0 modulator:

(i) induces a change in the fluorescence emission of at least one luminophore comprised in the composition or cell; and/or

(ii) induces a redistribution of the at least second polypeptide (B) and/or (D), and/or (iii) inhibits the translocation of the second polypeptide (B) and/or (D) together with the first

'.5 polypeptide (A) and/or (C) to the lipid bi-layer.

Further preferred is the method according to the invention, further comprising the step of synthesizing the identified modulator.

In a further aspect the invention provides the use of the method according to the invention for the identification of a test compound in a library of test compounds which

10 modulates a medically relevant protein-protein interaction. A preferred medically relevant protein-protein interaction is selected from the group consisting of a CaMKII subunit - a CaMKII subunit interaction; a CaMKII subunit - calmodulin interaction; an interaction in the NFkB complex; a p53 — MDM2 interaction; a p53 - p53 interaction; a CDK2 - cyclin Al interaction; a PIK3CA (pi 1 Oa) - PIK3R1 (p85a) interaction; a PIK3R1 (p85α) - IRSl

15 interaction; a Jun - Fos interaction; a Smad protein - Smad protein interaction; an interaction in the CDKNlA - PCNA - CDK2 - CCNAl complex; an interaction in the protein phosphatase 2 complex; an interaction in the HDACl-ETHE (HSCO)-TP53 complex; and a STAT3-STAT3 interaction. Preferably, the protein phosphatase A2 complex comprises the proteins PPP2R1 A,reg A (PR65); PPP2R5A, reg B (B56) and PPP2CA,cat (P2CA). Preferably, the NFkB

5 complex comprises the proteins RELA (p65); NFKBl (pi 05) and NFKBIA (IkB). It is also preferred that the CaMKII subunit - CaMKII subunit protein-protein interaction is selected from the group consisting of an CaMKII alpha - CaMKII alpha, CaMKII beta - CaMKII beta and CaMKII alpha - CaMKII beta interaction. In another preferred embodiment, the CaMKII - calmodulin protein-protein interaction is a CaMKII alpha - CaM and/or a CaMKII beta - CaM

0 interaction. Also preferred is the use according to the invention wherein the smad proteins are each individually selected from the group consisting of smadl, smad2, smad3, smad4 and ZFYVE9 (Sara).

Compounds that modulate medically relevant protein-protein can be used for the preparation of a medicament for the treatment of a disease selected from an inheritable disease,

5 cancer disease, viral diseases, organ failure, skin disease, allergy, bacterial disease, parasitic disease, fungal disease, a psychotic disorder and a prion disease. More preferably, said disease is selected from bladder cancer, melanoma, breast cancer, non-hodgkin's lymphoma, colon cancer, rectal cancer, pancreatic cancer, endometrial cancer, prostate cancer, kidney cancer, skin non- melanoma cancer, leukemia, thyroid cancer, lung cancer, aids, aids related complex, chickenpox,

!0 common cold, cytomegalovirus infection, Colorado tick fever, dengue fever, ebola haemorrhagic fever, hand, foot and mouth disease, hepatitis, bronchits, herpes simplex, herpes zoster, hpv, influenza, lassa fever, measles, marburg haemorrhagic fever, infectious mononucleosis, mumps, poliomyelitis, progressive multifocal leukencephalopathy, rabies, rubella, sars, smallpox, viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, west nile disease, yellow

>5 fever, anthrax, bacterial meningitis, botulism, brucellosis, campylobacteriosis, cat scratch disease, cholera, diphtheria, epidemic typhus, impetigo, legionellosis, leprosy, leptospirosis, listeriosis, lyme disease, melioidosis, mrsa infection, nocardiosis, pertussis, plague, pneumococcal pneumonia, psittacosis, q fever, rocky mountain spotted fever, salmonellosis, scarlet fever, shigellosis, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhoid fever,

50 typhus, urinary tract infections, african trypanosomiasis, amebiasis, ascariasis, babesiosis, chagas disease, clonorchiasis, cryptosporidiosis, cysticercosis, diphyllobothriasis, dracunculiasis, echinococcosis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, free-living amebic infection, giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, kala-azar, leishmaniasis, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, pinworm infection, scabies,

⁾5 schistosomiasis, taeniasis, toxocariasis, toxoplasmosis, trichinellosis, trichinosis, trichuriasis, trichomoniasis, trypanosomiasis, aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, tinea pedis, transmissible spongiform encephalopathy, bovine spongiform encephalopathy, creutzfeldt-jakob disease, kuru,fatal familial insomnia, alpers syndrome, atopic dermatitis, latex allergy, food allergy, acute interstitial nephritis, asthma,

5 diarrhoea, diabetes, actinic keratosis, angioma, athlete's foot, aquagenic pruritus, atopic dermatitis, baldness, basal cell carcinoma, bed sore, behcet's disease, blepharitis, boil, bowen's disease, bullous pemphigoid, canker sore, carbuncles, cellulitis, chloracne, chronic dermatitis of the hands and feet, cold sores, contact dermatitis, creeping eruption, dandruff, dermatitis, dermatitis herpetiformis, dermatofibroma, diaper rash, dyshidrosis, eczema, epidermolysis

0 bullosa, erysipelas, erythroderma, ferguson's disease, friction blister, genital wart, grover's disease, hidradenitis suppurativa, hyperhidrosis, ichthyosis, impetigo, jock itch, kaposi's sarcoma, keloid, keratoacanthoma, keratosis pilaris, lewandowsky-lutz dysplasia, lice infection, lichen planus, lichen simplex chronicus, lipoma, lymphadenitis, malignant melanoma, melasma, miliaria, molluscum contagiosum, nummular dermatitis, paget's disease of the nipple,

5 pediculosis, pemphigus, perioral dermatitis, photoallergy, photosensitivity, pityriasis rosea, pityriasis rubra pilaris, porphyria, psoriasis, raynaud's disease, ringworm, rosacea, scabies, scleroderma, sebaceous cyst, seborrheic keratosis, seborrhoeic dermatitis, shingles, skin cancer, skin tags, spider veins, squamous cell carcinoma, stasis dermatitis, tick bite, tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea pedis, tinea unguium, tinea versicolor, tinea, tungiasis,

O urticaria, schizophrenia and vitiligo.

CaMKII inhibitors are useful to treat heart failure, modulators of p53-MDM2, CDK2- cyclin Al and pl lθα-p85α protein-protein interactions are useful for the treatment of cancer. Modulators of protein-protein interactions within the NFKB multiprotein complex are useful to treat inflammation. Thus, especially preferred is a modulator isolatable from the method of the

'5 invention for the production of a medicament for the treatment of a disease selected from the group heart failure, cancer, inflammation and cystic fibrosis.

It is well known in the art that cellular assays can be used in high-throughput set ups, wherein the cells are incubated in multiple- well tissue culture plates, e.g. 6, 24, 96, 384 well- plates. The above described methods of the invention can also be used in high-throughput set ups

10 to identify potential binding partners or modulators of a given protein-protein interaction.

A further aspect of the invention is a method according to the invention further comprising the step of: (d) repeating steps (a) through (c) a plurality of times with a library that comprises a plurality of second polypeptides (B) that comprise different target polypeptides (d) and/or (f) or polynucleotides that encode such second polypeptides (B) for identifying target

15 polypeptides (d) and/or (f) which specifically bind to the bait polypeptide (b). In a preferred embodiment, the library is a combinatorial library.

A further aspect of the invention is a kit of parts comprising (i) at least a first expression vector comprising nucleic acids coding for a first polypeptide (A) of the invention except for the fact that in this specific expression vector the polynucleotide does not encode the bait

5 polypeptide (b) but is designed to allow in frame fusion to polynucleotides encoding the other components (a) and/or (c) as the case may be, e.g. a multiple cloning site (MCS), and (ii) at least a second expression vector comprising a polynucleotide encoding a fluorescent polypeptide which is designed to allow 5'- or 3'- in frame fusion to a polynucleotide encoding the target protein of choice. Preferred fluorescent polypeptides are described above. The MCS may be a

0 single restriction site or any multiple cloning site in the art and may be used by a skilled person to clone his or her bait and/or target polynucleotide into the expression vectors of the kit.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the 5 invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

The following figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in !θ any way.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 Panel A: Schematics of a composition comprising one first polypeptide (A) of the invention and a second polypeptide (B) of the invention. The bait polypeptide (b) of the

'.5 first polypeptide (A) in this example is bound to the first target polypeptide (d) of the second polypeptide (B) via a non-covalent interaction. LB indicates the lipid bi-layer on the left hand side of the figure to which is bound the recruiting polypeptide (a). Panel B: Schematic structure of preferred vectors allowing C-terminal fusion of a bait polypeptide to an Annexin A4-fluorescent protein fusion (pA4-FP-Cl) or C-terminal

10 fusion of a bait polypeptide to an Annexin A4-fluorescent protein fusion (pFP-A4-Nl) and schematic structure of preferred vectors allowing C-terminal fusion of a target polypeptide to a fluorescent protein (pFP-Cl) and N-terminal fusion of a target polypeptide to a fluorescent protein (pFP-Nl). Fig. 2 Alignment of various annexin proteins. Each annexin core domain is indicated by bold- letter amino acids.

Fig. 3A Annexin A4 localization and translocation in living cells. (A) Schematic view of

5 annexin A4 cellular distribution. The protein is localized in the cytoplasm and in the nucleus. Following a rise in intracellular calcium concentration, the protein binds to the lipid bi-layer which is the plasma and the nuclear membrane. (B) EYFP-annexin A4 expressed in NlE-115 cells translocates to cell membranes upon calcium elevation with ionomycin. (C) The graph shows protein distribution across cells. White bar indicates

0 position of the measurement.

Fig. 3B through 3D: Localization and translocation of various fluorescent protein-target polypeptide fusion (left hand pictures) and annexin A4-fluorescent protein-bait polypeptide fusions (right hand pictures). Images of cells (NlE-115, unless stated 5 otherwise) transfected with constructs, a) Target fusion proteins show no translocation following ionomycin addition, b) Bait fusion proteins translocate in response to calcium. All scale bars are lOμm.

Fig. 4 Annexin-based protein-protein interaction assay. (A) Schematic view of the assay. The

!0 bait protein is fused to a fluorescent protein (FP) and annexin A4. The target protein is fused to a complementary FP. The two constructs are expressed in cytoplasm and/or in the nucleus of cells. Increasing calcium concentration leads to annexin-FP-bait translocation to the plasma membrane and/or the nuclear membrane. Target-FP translocates as well only in case the bait and the target polypeptide interact. (B) An

Ϊ5 example of a protein-protein interaction visualized using annexin-based assay. PI3 kinase is composed of a catalytic (PIK3CA (pi 1 Oa)) and a regulatory subunitPIK3Rl

(p85α)). Annexin A4-ECFP-p85α fusion is used as the bait. EYFP-pl 10a is used as the target. The constructs are co-transfected in NlE-1 15 cells. They localize in the cytoplasm of unstimulated cells. Upon stimulation of cells with ionomycin, both the

10 target and the bait translocate to the plasma membrane, indicating their interaction. (C)

The graphs show protein distribution across cells. White bars indicate position of the measurement. The graphs further illustrate a similar change in distribution of both the bait and the target. Fig. 5 Protein complex analysis using annexin-based assay. (A) Schematic view of the assay. The bait protein is fused to a fluorescent protein and annexin A4. Up to three additional proteins can be fused to complementary fluorescent proteins and visualized simultaneously in living cells. Based on the translocation of different fluorescent

5 proteins, it can be determined which components of the complex interact at any given moment. (B) A three-component complex analysis. NFKB complex composed of RELA (p65), NFKBl (plO5) and NFKBIA (IKB) was visualized in NlE-115 cells. Annexin A4-ECFP-ρ65fusion is used as the bait. EGFP-IKB and EYFP-plO5 are used as targets. Translocation of all three fusions is observed upon ionomycin addition indicating

0 complex formation. (C) The graphs show protein distribution across cells. White bars indicate position of the measurement. The graphs further illustrate a similar change in distribution of the bait and both targets.

Fig. 6 Additional examples of protein-protein interactions and protein complexes analyzed

5 using annexin-based assay in NlE-115 cells. (A) TP-53 - MDM2 interaction. TP53-

ECFP-annexin A4 fusion is used as the bait. MDM2-EYFP is the target. Translocation of both bait and the target indicates interaction. (B) CDK2 - CCNAl (cyclin Al) interaction. Annexin A4-ECFP-CDK2 fusion is used as the bait. EYFP-cyclin Al is the target. Translocation of both bait and the target indicates interaction. (C) CaMKIIβ-

>0 CaMKIIβ-calmodulin complex. Annexin A4-EYFP-calmodulinfusion is used as the bait. ECFP-CaMKIIa and EGFP-CaMKIIβ are used as targets. Translocation of all three fusions is observed upon ionomycin addition indicating complex formation.

Fig. 7 Analysis of the hirachy of interactions in the CDK2-PCNA-CCNAl-p21 complex.

>5

Fig. 8 Effect of small molecule protein-protein interaction inhibitors measured using annexin- based assay. (A) NlE-115 cells are shown expressing A4-EYFP-TP53 (bait) and ECFP- MDM2 (target). Ionomycin addition elevates intracellular calcium levels causing translocation of both annexin-labeled TP53 and MDM2 due to TP53-MDM2

10 interaction. Subsequent cell treatment with nutlin-3, TP53-MDM2 interaction inhibitor, results in partial relocation of MDM2 from cellular membranes back to the cytosol. (B) The graphs show protein distribution across cells. White bars indicate position of the measurement. The graphs further demonstrate the effect of the drug on MDM2 distribution.

55 EXAMPLES

Example 1: Construction of Expression Plasmids

DNA expression vectors comprising polynucleotides encoding the polypeptides of the 5 first polypeptide (A) and/or polypeptides comprised in the composition of the invention can be generated by standard molecular cloning techniques well known to the artisan of molecular cloning.

In the following, it will be outlined how to clone annexin A4 into some exemplary expression constructs. This serves as a general guideline of how to clone proteins of the annexin 0 family.

The cDNA for human annexin A4 was obtained from LGC Promochem (Wesel,

Germany). To construct the ECFP-annexin A4 and EYFP-annexin A4 fusions, the coding sequence of annexin A4 was amplified by PCR. The resulting product was digested with EcoPJ and BamHI and inserted into the pE YFP-Cl and pECFP-Cl vectors (Clontech, Palo Alto, CA;

5 ECFP had two additional mutations, as described in Llopis et al., 2000). To generate the annexin

A4-ECFP and annexin A4-EYFP fusions, annexin A4 was amplified by PCR. The product was digested with EcoRI and BamHI and ligated into pEYFP-Nl (SEQ ID NO: 23) and pECFP-Nl

(SEQ ID NO: 22) vectors (Clontech). To construct the EYFP-annexin A4 core fusion, the sequence encoding the core of annexin A4 was amplified by PCR. The resulting product was

!0 digested with EcoRI and BamHI and inserted into the pEYFP-Cl vector (Clontech).

The vectors generated along the lines outlined above were termed pA4-ECFP-Cl (SEQ ID NO: 24) and pEYFP-A4-Nl (SEQ ID NO: 25)

Example 2: Construction of Bait and Target Fusion plasmids

»5 pi 10a (PIK3CA; RZPD: IOH36320) was amplified by PCR and inserted in pA4-EYFP-

Cl vectors using HindIII and Kpnl. p85α (PIK3R1; Origene: TCl 15320) was inserted into pA4- ECFP-Cl using EcoRI and Sail. IRSl (Origene: TC124032) was inserted into pA4-mCherry-Cl using Sad and Sail. p65 (RELA) was obtained from J. Schmid (Vienna) as pmDsRed-Cl-p65 and subcloned

JO into pA4-ECFP-Cl vector using HindIII and BamHI. pl05 (NFKBl ; RZPD: IRATp970B1075D) was inserted into pA4-E YFP-Cl and pEYFP-A4-Nl vectors using HindIII and Kpnl. p50 (aa 1- 433 of pi 05) was inserted into pA4-EYFP-Cl and pA4-mCherry-Cl using HindIII and Kpnl. IDB (NFKBIA; RZPD: IRAUp969B0119D) was inserted into pA4-EGFP-Cl and pA4- mCherry-Cl using HindIII and Kpnl. PP lα (PPPlCA; Origene: TC 127915) was inserted into pA4-EYFP-Cl using HindIII and Kpnl. PPlγ (PPPlCC) was a kind gift from R. Russell (EMBL Heidelberg). The cDNA was amplified by PCR and inserted into pA4-E YFP-Cl using HindIII and Kpnl. Inhibitor- 1 (PPPlRlA; Origene: TCl 15900) was inserted into pA4-)ECFP-Cl using HindIII and Kpnl. 5 Inhibitor-2 (PPP1R2; Origene: TCl 16238) was inserted into pA4-ECFP-Cl using Sad and Kpnl.

Rat CaMKIIa and CaMKIIβ was obtained from T. Meyer (Stanford) as pEGFP-Cl-

CaMKIIa and pEGFP-Cl -CaMKIIβ. pECFP/E YFP-Cl -CaMKIIβ were created by subcloning of

ECFP/EYFP from pECFP/E YFP-Cl into the above mentioned EGFP vectors using Nhel and

0 BgIII. pECFP/EYFP-Cl-CaMKIIα were created by subcloning of ECFP/EYFP from pECFP/EYFP-Cl into the corresponding EGFP vectors using Nhel and HindIII. pA4-

ECFP/EYFP-Cl-CaMKIIα/β were created by inserting PCR amplified annexin A4 into pECFP/EYFP-Cl-CaMKIIα/β using Nhel/Agel. Calmodulin (RZPD: IRALp962F191Q2) was amplified by PCR and inserted into pE YFP-Cl using HindIII and BamHI to create pEYFP-Cl-

5 calmodulin. pA4-EYFP-calmodulin was created by inserting PCR amplified annexin A4 into pEYFP-Cl -calmodulin using Nhel/Agel.

CDK2 (Origene: TC 109060) was inserted into pA4-ECFP-Cl using EcoRI and Sail. Cyclin Al (CCNAl; Origene: TC127871) was inserted into pA4-EYFP-Cl using HindIII and Kpnl. p21 (CDKNlA; Origene: SCl 19947) was inserted into pA4-EGFP-Cl using HindIII and 10 Kpnl. PCNA (Origene: SCl 18528) was inserted into pA4-mCherry-Cl using Sad and SacII. p53 (TP53; Origene: TCl 19832) was inserted into pA4-ECFP-Cl, pA4-EYFP-Cl, pA4- mCherry-Cl and pECFP(-A4)-Nl using HindIII and Kpnl. MDM2 (Origene: TCl 18660) was inserted into pA4-ECFP-Cl, pA4-EYFP-Cl and pEYFP(-A4)-Nl using Sad and SacII.

SH3 domain of AbIl kinase (ABLl; a kind gift of R. Russell, EMBL Heidelberg) was '.5 amplified by PCR and inserted into pA4-ECFP-Cl using Sad and SacII restriction enzymes. pA4-ECFP-Cl-FWLpeptide (FWL-peptide: QETFSDLWKLLPEN; SEQ ID: 26) and pECFP-Cl-APTpeptide (APT-peptide: APTYSPPPPP, SEQ ID NO: 27) were generated in the following way. Primers were designed that encode the desired sequence flanked with HindIII and Kpnl restriction sites. The primers were 5'-phosphorylated with the T4 polynucleotide •0 kinase (NEB) according to the manufacturer's instructions. The primers were heated to 95°C for 5 min and cooled down to anneal. Such double-stranded primers were ligated into the pA4- EYFP-Cl and pECFP-Cl vectors digested with HindIII and Kpnl restriction enzymes.

Three specific examples of expression vectors generated as outlined above and used in the invention are provided in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:21. SEQ ID NO:

15 16 comprises a polynucleotide sequences encoding a fusion protein comprising in N- to C- terminal order: the annexin A4 protein, the ECFP protein and the p85α protein, SEQ ID NO: 17 comprises a polynucleotide sequence encoding a fusion protein comprising in N- to C-terminal order: the EYFP protein and the pi 10a and SEQ ID NO:21 is identical to SEQ ID NO:16 but for a modified linker 1 encoding sequence, which is one codon shorter, i.e. the linker of the encoded 5 fusion protein is one amino acid shorter.

Example 3: Cell Culture and Transfection

HeLa and NlE-115 cells were passaged and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 0.1 mg/ml primocin. MDCK2 cells were maintained in MEM

0 supplemented with 5% FBS, 2 mM L-glutamine, and 0.1 mg/ml primocin. For imaging experiments, cells were plated in 35-mm MatTek chambers (Ashland, MA) and transfected at 50-70% confluency. HeLa and NlE-115 cells were transfected with FuGENE 6 reagent (Roche, Mannheim, Germany). MDCK2 cells were transfected with either FuGENE 6 or Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Transfections were performed in Opti-MEM (Invitrogen)

5 according to the manufacturer's instructions. For multiple transfections, equal amounts of DNA were used for each sensor. Cells were washed 12-24 h after transfection and incubated in imaging medium (20 mM HEPES, pH 7.4, 115 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM K₂HPO₄, 2 g/1 D-glucose) at 37°C with 5% CO₂ for between 20 and 30 min before imaging. A DMSO stock of ionomycin (Calbiochem) was prepared and ionomycin prediluted in imaging

!0 medium before it was carefully added to the dish to induce translocation. To induce relocation of the fusion proteins 5 μM ionomycin were added to the cell culture.

Example 4: Imaging and image analysis

>5 Equipment and settings

All experiments were performed on a Leica TCS SP2 AOBS microscope (Leica Microsystems).

An HCX PL APO lbd.BL 63.Ox 1.40 oil objective was used. Pinhole was half-opened in all experiments (2.62 airy). Laser power and PMT gain were adjusted from experiment to experiment. Images were taken in 8 bit mode, with 2-4 line averaging. SO General excitation and emission settings are as follows:

• ECFP excitation 405 nm emission 465-495 nm

• EGFP excitation 488 nm emission 495-510 nm

• EYFP excitation 532 nm emission 545-570 nm • mCherry excitation 594 nm emission 605-650 nm

Alternatively, emission settings can be modified. For instance, also 458 nm laser can be used for ECFP excitation, 515 nm for EYFP excitation and 561 nm laser for mCherry excitation. Emission setting can be modified depending on the excitation settings and can be 450-480 nm for ECFP, or 530-570 nm for EYFP. The optimal excitation and emission settings depend on the fluorescent protein combination used in a particular experiment.

Image processing

All image processing and calculations were performed using ImageJ (http://rsb.info.nih.gov/ij/). Background level was measured outside cells and subtracted globally. Median filter (1-2 pixel) was used for image smoothening. Profile graphs were obtained by measuring fluorescence signal across cells where it is indicated by the white line.

Example 5: Interaction pairs

Plasmids coding for polypeptides comprised in the composition according to the present invention were transfected into eukaryotic cells. A summary of interactions analyzed using the annexin-based assay of the invention using different proteins as baits/targets is shown in Table 1 (each bait- and target polypeptide is indicated in bold letters). The protein-protein interaction between all of the medically relevant proteins shown in Table 1 were measured using the methods of the invention and the binding between the respective proteins was observed unless specifically indicated (see also Figures 1-6). In all experiments N-terminal fused bait polypeptides or target polypeptides were used unless indicated otherwise.

TABLE 1

TABLE 1 (CONTINUED)

Interaction: SH3-APT peptide

A4-ECFP-SH3 ECFP-APT pep Interaction of AbIl kinase with poly praline peptide

Interaction : PPP2R1 A,reg A (PR65) - PPP2R5A, reg B (B56) - PPP2CA,cat (P2CA)

A4-EYFP-PPP2R1A ECFP-PPP2R5A Interaction of the 2 regulatory subunits of the protein phosphatase 2

Example 6: Hierarchical analysis of Interaction pairs

In most two-component protein complex analysis experiments ECFP and EYFP were used as fluorescent protein labels. The emission and excitation settings were: ECFP ex. 458 run, em. 465-495 nm; EYFP ex. 515 ran, em. 530-550 ran. In order to study larger complexes, additional fluorescent proteins were introduced. The third component was typically labeled with mCherry and the fourth with EGFP. The imaging settings in these experiments were as follows: ECFP ex. 405 nm, em. 450-480 nm; EGFP ex. 488 nm, em. 495-510 nm; EYFP ex. 532 nm, em. 545-565 nm; mCherry ex. 594 nm, em. 605-650 nm. The use of these fluorescent proteins and settings enabled the analysis of four-component complex. For this purpose a known complex between CDK2, cyclin Al (CCNAl), p21 (CDKNlA) and PCNA and prepared bait and target constructs as described above were used. A series of experiments was performed to determine if complex formation can be observed in a way that would be expected from the literature, according to which interaction between CDK2-cyclin Al complex and PCNA is mediated by p21 protein. CDK2 was used as bait and its interaction with cyclin Al was first demonstrated (see Figure 7). PCNA was then added to the system and an interaction between CDK2 and cyclin Al was observed as before but no interaction with PCNA. If p21 was used in combination with CDK2 and PCNA, and cyclin Al was left out, translocation of both targets was observed which confirms the bridging function of p21. When all components were introduced in a single cell, formation of the entire four protein complex was observed. Finally, p21 was used as bait and its interaction with all components of the complex individually, including interaction with cyclin Al, and all possible combinations was confirmed (Table 2). The experiments demonstrate that the hierarchy of the complex is (CDK2-cyclin A 1 )-p21 -(PCNA).

Table 2

Example 7: Small molecule disruption of protein-protein interaction

The known p53-MDM2 interaction inhibitor nutlin-3 was used to interfere with the protein-protein interaction between p53 and MDM2 and partial reversion of translocation of the target was achieved while leaving the bait unaffected (Figure 8). The experiment evidences the possibility to use the methods of the invention in detecting inhibitors of protein-protein interactions in high-throughput set-ups.

Claims

1. A first polypeptide (A) comprising, essentially consisting or consisting of:

(i) a recruiting polypeptide (a) comprising, essentially consisting or consisting of at 5 least an annexin core domain or a functional variant thereof;

(ii) a bait polypeptide (b); and (iii) a luminophore (c).

2. The first polypeptide (A) according to claim 1, wherein the annexin core domain is from 0 an annexin selected from the group consisting of annexin Al , A2, A4, A5 and A6.

3. The first polypeptide (A) according to claims 1 or 2, wherein the annexin core domain comprises at least an amino acid sequence according to SEQ ID NOs: 1-5.

5 4. The first polypeptide (A) according to claim 1, wherein the functional variant of an annexin core domain is: (i) a polypeptide having at least 60% similarity over the entire length of an annexin core domain according to SEQ ID NOs: 1-5, or

(ii) a polypeptide according to (i) having N-, C- and/or internal deletions or insertions. »0

5. The first polypeptide (A) according to claim 1, wherein the recruiting polypeptide (a) comprises, essentially consists, or consists of an annexin selected from the group consisting of annexin Al, A2, A4, A5 and A6.

55 6. The first polypeptide (A) according to claim 5, wherein the annexin has an amino acid sequence according to SEQ ID NOs: 11-15.

7. The first polypeptide (A) according to any of claims 1 to 6, wherein the bait polypeptide (b) is selected from the group consisting of an antibody; an enzyme, in particular a iO kinase or a phosphatase; a receptor; a DNA-binding protein; an apoptosis- related protein; a DNA synthesis protein; a DNA repair protein; a receptor; a transcription factor; a cell cycle regulator; a lipid-binding protein; a lectin; a cytokine; a serpin; a cell-surface antigen; a growth factor; a heat-shock protein; a hormone; a PDZ-domain harbouring protein; a viral protein and a bacterial protein or fragments thereof.

8. The first polypeptide (A) according to claim 7 wherein the bait polypeptide (b) is selected from the group consisting of CaMKIIa; CaMKIIβ; Calmodulin; p53; MDM2; RELA (p65); NFKBI (plO5); NFKBIA (IKB); CDK2; CDK4; CCNAl (cyclin Al);

5 PIK3CA (pl lOα) and PIK3R1 (p85α); insulin receptor substrate 1 (IRSl); jun; fos; smad 1; smad2; smad3; smad4; ZFYVE9; CDKNlA; PCNA; HDACl; ETHE (HSCO); STAT3; PPP2R1 A,reg A (PR65); PPP2R5A,reg B (B56) and PPP2CA, cat (P2CA).

9. The first polypeptide (A) according to any of claims 1 to 8, wherein the luminophore (c) 0 is a polypeptid, a nano fluorescent particle (NFP) and/or a fluorescent dye.

10. The first polypeptide (A) according to claim 9, wherein the polypeptide comprises at least a functional fragment of a protein that is selected from the group consisting of GFP, EGFP, ECFP, BFP, EYFP, CFP, dsRED, dsRED2, mCherry, mPlum, luciferase,

5 horseradish peroxidase and variants thereof.

11. The first polypeptide (A) according to claims 9 to 10, wherein the luminophore (c) is a polypeptide and the polypeptides (a) (b) and (c) in said first polypeptide (A) are fused to each other, optionally via one or two linkers, from N- to C-terminus in the order (a)-(b)-

^•0 (C), (a)-(c)-(b), (b)-(a)-(c), (b)-(c)-(a), (c)-(a)-(b) or (c)-(b)-(a).

12. The first polypeptide (A) according to claims 1 to 11, wherein the first polypeptide does not comprise a nuclear localization signal (NLS) sequence.

Ϊ5 13. A composition comprising at least one first polypeptide (A) according to claims 1 to 12, and further comprising at least a second polypeptide (B) comprising, essentially consisting, or consisting of:

(i) a first target polypeptide (d); and

(ii) a luminophore (e). 10

14. The composition of claim 13, wherein the first target polypeptide (d) is identical or different to the bait polypeptide (b).

15. The composition of claims 13 or 14, wherein the composition further comprises one or more further first polypeptides (C).

16. The composition of any of claims 13 to 15, wherein the composition further comprises one or more further second polypeptides (D).

17. The composition of any of claims 13 to 16, wherein the composition further comprises one or more third polypeptides (E) comprising, essentially consisting or consisting of a target protein (f) without luminophore.

18. The composition of any of claims 13 to 17, wherein the luminophore (e) is a polypeptid, a nano fluorescent particle (NFP) and/or a fluorescent dye.

19. The composition of any of claims 13 to 18, wherein at least the luminophore (c) is different from luminophore (e).

20. The composition according to any of claims 13 to 19, wherein at least two of the luminophores are capable of fluorescence resonance energy transfer (FRET).

21. The composition according to any of claims 13 to 20, wherein the bait polypeptide (b) is capable of specifically binding to the one or more target protein (d) and/or (f).

22. The composition according to claims 13 to 21, wherein the target protein (d) and/or (f) is each individually selected from the group consisting of CaMKIIa; CaMKIIβ; Calmodulin; p53; MDM2; RELA (p65); NFKBI (pi 05); NFKBIA (IKB); CDK2; CDK4;

CCNAl (cyclin Al); PIK3CA (pi 10a) and PIK3R1 (p85α); insulin receptor substrate 1

(IRSl); jun; fos; smad 1; smad2; smad3; smad4; ZFYVE9; CDKNlA; PCNA; HDACl;

ETHE (HSCO); STAT3; PPP2R1 A,reg A (PR65); PPP2R5A,reg B (B56) and PPP2CA, cat (P2CA).

23. The composition according to claims 13 to 22, wherein the composition further comprises a lipid bi-layer.

24. The composition of claim 23, wherein the lipid bi-layer is a plasma-membrane of a cell, the nuclear-membrane of a cell, the membrane of the endoplasmic reticulum of a cell, a liposome and/or a lipid membrane on a solid support.

5 25. A polynucleotide encoding the first polypeptide (A) in any of claims 1-12.

26. An expression vector comprising the polynucleotide of claim 25 and optionally one or more polynucleotides encoding one or more second polypeptide (B), one or more further first polypeptide (C), one or more of the further second polypeptide (D) and/or

0 one or more third polypeptide (E).

27. A cell comprising a polynucleotide according to claim 25, and/or an expression vector according to claim 26 and optionally one or more polynucleotides encoding one or more second polypeptide (B), one or more further first polypeptide (C), one or more further

5 second polypeptide (D) and/or one or more third polypeptide (E) or expression vectors comprising these one or more polynucleotides.

28. The cell according to claim 27, wherein said cell is a eukaryotic cell.

10 29. A method of detecting a protein-protein interaction, comprising the steps:

(a) providing a composition according to any of claims 23 to 24 or a cell according to claims 27 to 28;

>5 (c) and detecting at least one luminophore (c) and/or (e).

30. The method according to claim 29, wherein the lipid bi-layer is permeabilized before, during or after step (b).

50 31. The method according to claim 29 or 30, wherein step (b) comprises the contacting of the composition or the cell with a solution comprising a substance that increases the Ca²⁺ ion concentration in comparison with the Ca²⁺ ion concentration in the composition or the cell in step (a).

32. The method according to claim 31 wherein the substance is selected from the group consisting of Ca²⁺, in particular CaCl_2, calcium lactate, calcium gluconate, calcium malate, ionomycin, calcium ionophore A23187, calcium ionophore 8-Br-A23187 and thapsigarin.

5

33. The method according to any of claims 29 to 32, wherein step (c) is carried out before, during and/or after step (b).

34. The method according to any of claims 29 to 33, wherein the at least one luminophore is 0 detected by measuring the fluorescence emission.

35. The method according to any of claims 29 to 34, wherein in step (c) at least one luminophore is detected, which is bound to the lipid bi-layer.

5 36. The method according to any of claims 29 to 35, wherein in step (c) at least one luminophore is detected, which is not bound to the lipid bi-layer.

37. The method according to any of claims 29 to 36, wherein the detection is carried out by utilizing a device selected from the group consisting of a fluorescent plate reader, a

!0 fluorescence activated cell sorting (FACS) apparatus, a photomultiplier tube, a linear diode array, a video camera and a charge-coupled device (CCD).

38. A method of identifying a modulator of protein-protein interaction comprising the steps of the method of any of claims 29 to 37, and further comprising the step of contacting

15 the composition or the cell with a test compound.

39. The method according to claim 38, wherein the contacting occurs prior, concomitantly or after steps (a), (b) or (c).

10 40. The method according to claims 38 or 39, wherein the modulator:

(i) induces a change in the fluorescence emission of at least one luminophore comprised in the composition or cell; and/or (ii) induces a redistribution of the at least second polypeptide (B) and/or (D), and/or (iii) inhibits the translocation of the second polypeptide (B) and/or (D) together with the first polypeptide (A) and/or (C) to the lipid bi-layer.

41. The method of any of claims 38 to 40, further comprising the step of synthesizing the 5 identified modulator.

42. Use of the method according to claim 38 to 40, for the identification of a test compound in a library of test compounds which modulates a medically relevant protein-protein interaction.

0

43. Use according to claim 42 wherein the medically relevant protein-protein interaction is selected from the group consisting of a CaMKII subunit - a CaMKII subunit interaction; a CaMKII subunit - calmodulin interaction; an interaction in the NFkB complex; a p53 - MDM2 interaction; a p53 - p53 interaction; a CDK2 - cyclin Al

5 interaction; a PIK3CA (pi 10a) - PIK3R1 (p85α) interaction; a PIK3R1 (p85α) - IRSl interaction; a Jun - Fos interaction; a Smad protein - Smad protein interaction; an interaction in the CDKNlA - PCNA - CDK2 - CCNAl complex; an interaction in the protein phosphatase 2 complex; an interaction in the HDACl-ETHE (HSCO)-TP53 complex; and a STAT3-STAT3 interaction.

^•0

44. Use according to claim 43 wherein the protein phosphatase A2 complex comprises the proteins PPP2R1 A,reg A (PR65); PPP2R5A, reg B (B56) and PPP2CA,cat (P2CA).

45. Use according to claim 43 wherein the NFkB complex comprises the proteins RELA !5 (p65); NFKBl (pi 05) and NFKBIA (IkB).

46. Use according to claim 43, wherein the CaMKII subunit - CaMKII subunit protein- protein interaction is selected from the group consisting of an CaMKII alpha - CaMKII alpha, CaMKII beta - CaMKII beta and CaMKII alpha - CaMKII beta interaction.

10

47. Use according to claim 43, wherein the CaMKII - calmodulin protein-protein interaction is a CaMKII alpha - CaM and/or a CaMKII beta - CaM interaction.

48. Use according to claim 43, wherein the Smad proteins are each individually selected from the group consisting of smadl, smad2, smad3, smad4 and ZFYVE9 (Sara).

49. A method according to any of claims 18-31, further comprising the step of : (d) repeating steps (a) through (c) a plurality of times with a library that comprises a plurality of second polypeptides (B) that comprise different target polypeptides (d) and/or (f) or polynucleotides that encode such second polypeptides (B) for identifying target polypeptides (d) and/or (f) which specifically bind to the bait polypeptide (b).

50. The method according to claim 49, wherein said library is a combinatorial library.