WO1997010502A1

WO1997010502A1 - A high throughput assay using fusion proteins

Info

Publication number: WO1997010502A1
Application number: PCT/US1996/014563
Authority: WO
Inventors: Scott P. Salowe
Original assignee: Merck & Co., Inc.
Priority date: 1995-09-15
Filing date: 1996-09-11
Publication date: 1997-03-20
Also published as: EP0855029A4; EP0855029A1; CA2231385A1; JP2000501171A

Abstract

This application describes a high throughtput assay for screening for compounds which are capable of binding to a fusion protein which consists of a target protein and an FK506-binding protein.

Description

TITLE OF THE INVENTION

A HIGH THROUGHPUT ASSAY USING FUSION PROTEINS

BACKGROUND OF THE INVENTION Src homology 2 (SH2) domains are a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts. They have routinely been expressed in E. coli as fusion proteins with glutathione-S- transferase (GST). This usually provides high level expression and straightforward affinity purification on glutathione-Sepharose. Ligand binding is then assayed by incubating the GST/SH2 with a radiolabeled phosphopeptide, precipitating the complex with glutathione-Sepharose, washing the beads, and then counting the beads to determine bound radioactivity [Isakov et al., J. Exp. Med., 181 , 375-380 (1995); Piccione et al., Biochemistry, 32, 3197-3202 (1993); Huyer et al., Biochemistry, 34, 1040-1049 (1995)]. There are several disadvantages to this procedure, particularly when applied to high-throughput screening for agonists, antagonists, or inhibitors as new leads for drug development. First, the radiolabeling of the peptide is carried out either enzymatically with a kinase and [32p]ATP or chemically with [ 125τ] Bolton-Hunter reagent. In both cases, the isotopes are short-lived and thus require frequent preparation of material. In the case of enzymatic phosphorylation, the appropriate kinase must also be available in sufficient quantity to generate enough material for screening purposes. Second, the protocol requires separation of bound complex from free phosphopeptide by washing of the glutathione-Sepharose beads. This is a nonequilibrium procedure that risks dissociation of the bound ligand, particularly when off-rates are fast. Thus, there is the possibility of misleading results. Finally, due to the number of manipulations and centrifugations involved, the protocol is very tedious to conduct manually and is not readily adaptable to robotic automation to increase throughput.

Two additional methods for measuring the interaction of proteins and ligands that have been applied to SH2 domains are biospecific interaction analysis using surface plasmon resonance and isothermal titration calorimetry (Felder et al., Mol. Cell. Biol., 13, 1449- 1455 (1993); Panayotou et al., Mol. Cell. Biol., 13, 3567-3576 (1993); Payne et al., Proc. Natl. Acad. Sci. U.S A., 90, 4902-4906 (1993); Morelock et al., J. Med. Chem. 38, 1309-18 (1995); Ladbury et al., Proc. Natl. Acad. Sci. U.S. A., 92, 3199-3203 (1995); Lernmon et al.,

Biochemistry, 33, 5070-5076 (1994)). These techniques do not require a particular fusion partner for the SH2 domain, but do require sophisticated instrumentation that is not amenable to high throughput screening.

SUMMARY OF THE INVENTION

The instant invention covers a method of screening for compounds capable of binding to a fusion protein which comprises combining a test compound, a tagged ligand, a fusion protein (target protein, peptide linker and FK506-binding protein), a radiolabeled ligand, and coated scintillation proximity assay (SPA) beads, and then measuring the scintillation counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a control assay in the absence of the test compound, so as to determine the effect the test compound has on the binding of the tagged ligand. This invention provides an immediate means of making use of SPA technology for the functional assay of ligand binding to a single or multiple signal transduction domain(s), for example a phosphopeptide binding to an SH2 domain. The present invention does not require specialized radiochemical synthesis and is readily adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors. BRIEF DESCRIPΗON OF THE FIGURES Figure 1.

A.) Binding of the streptavidin SPA bead, biotinylated ligand and the fusion protein (SH2:FKBP), which emits a detectable signal; and B.) Binding of the test compound and the fusion protein (SH2:FKBP), which results in no signal detection .

DETAILED DESCRIPΗON OF THE INVENTION

T e present invention relates to a method of screening for compounds which preferentially bind to a target protein.

An embodiment of this invention is a method of screening for compounds capable of binding to a fusion protein which comprises the steps of: a) mixing a test compound, a tagged ligand, the fusion protein, a radiolabeled ligand and coated scintillation proximity assay

(SPA) beads; b) incubating the mixture for between about 1 hour and about 24 hours; c) measuring the SPA bead-bound counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound using scintillation counting; and d) determining the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a control assay run in the absence of the test compound.

The term "fusion protein" refers to a "target protein" fused to an "FK506-binding protein" (FKBP), the two proteins being separated by a "peptide linker". A "peptide linker" may consist of a sequence containing from about 1 to about 20 amino acids, which may or may not include the sequence for a protease cleavage site. An example of a peptide linker which is a protease cleavage site is represented by the amino acid sequence GLPRGS. The term "target protein" refers to any protein that has a defined ligand. Included within this definition of target protein are single and multiple signal transduction domains, such as, but not limited to, Src homology 1 (SHI), Src homology 2 (SH2), Src homology 3 (SH3), and pleckstrin homology (PH) domains [Hanks & Hunter, FASEB J., 9, 576- 596 (1995); Bolen, Curr. Opin. Immunol., 1, 306-31 1 (1995); Kuriyan & Cowburn, Curr. Opin. Struct. Biol., 3, 828-837 (1993); Cohen et al., Cell, 80, 237-248 (1995)]. The term "SHI domain" refers to a family of homologous protein domains that bind ATP and catalyze tyrosine phosphorylation of peptide and protein substrates. The term "SH2 domain" refers to a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts. The term "SH3 domain" refers to a family of homologous protein domains that share the common property of recognizing polyproline type II helices. The term "PH domain" refers to a family of homologous protein domains that mediate both protein- protein and protein-lipid interactions. Examples of SH2 domains which may be utilized in the method of the invention include, but are not limited to, the single and tandem SH2 domains present in the tyrosine kinases ZAP, SYK and LCK. The DNA sequences were obtained from

GenBank, National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894. The Accession Numbers for the sequences are: human ZAP (L05148); human SYK (L28824) and human LCK (XI 3529). The term "tagged ligand" refers to a biotinylated or epitope tagged ligand for the target protein.

The term "radiolabeled ligand" refers to a [³H]- or [1²⁵I]- labeled ligand which binds to the FKBP. An example of a radiolabeled ligand useful in the instant invention is [3H]-dihydroFK506. The term "coated scintillation proximity assay beads" (SPA beads) refers to streptavi din-coated scintillation proximity assay beads when the tagged ligand is biotinylated, and to anti-epitope antibody bound to anti-antibody-coated or protein A-coated scintillation proximity assay beads when the tagged ligand is epitope-tagged. The term "control assay" refers to the assay when performed in the presence of the tagged ligand, the fusion protein, the radiolabeled ligand and the coated scintillation proximity assay beads, but in the absence of the test compound.

The term FK506-binding proteins may include, but are not limited to, the below listed FKBPs and FKBP homologues, which include a citation to the references which disclose them. This list is not intended to limit the scope of the invention.

Mammalian FKBP- 12 Galat et al., Eur. J. Biochem., 216:689-

707 (1993).

FKBP- 12.6 Wiederrecht, G. and F. Etzkorn

Perspectives in Drug Discovery and

Design , 2:57-84 (1994).

FKBP- 13 Galat et al., supra; Wiederrecht and

Etzkorn, supra.

FKBP-25 Galat et al., supra; Wiederrecht and

Etzkorn, supra. FKBP-38 Wiederrecht and Etzkorn, supra. FKBP-51 Baughman et al., Mol. Cell. Biol., 8,

4395-4402(1995) .

FKBP-52 Galat et al., supra.

Bacteria

Legionella pneumophilia Galat et al., supra. Legionella micadei Galat et al., supra. Chlamydia trachomatis Galat et al., supra. E. coli fkpa Home, S.M. and K.D. Young, Arch.

Microbiol., 163:357-365 (1995).

E. coli slyD Roof et al., J. Biol. Chem. 269:2902-2910

(1994).

E. coli orfl49 Trandinh et al., FASEB J. 6:3410-3420

(1992). Neisseria meningitidis Hacker, J. and G. Fischer, Mol. Micro.,

10:445-456 (1993).

Streptomyces chrysomallus Hacker and Fischer, supra.

Fungal yeast FKBP- 12 Cardenas et al., Perspectives in Drug

Discovery and Design , 2:103-126

(1994). yeast FKBP- 13 Cardenas et al., supra. yeast NPR 1(FPR3) Cardenas et al., supra. Neurospora Galat et al., supra.

A variety of host cells may be used in this invention, which include, but are not limited to, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.

Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express genes in a variety of host cells, such as, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.

Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Commercially available vectors suitable for FKBP fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pT7 (USB), pET (Novagen), pIBI (IBI), pProEX-1 (Gibco/BRL), pBluescript II (Stratagene), pTZ18R and pTZ19R (USB), pSE420 (Invitrogen), pVL1392 (Invitrogen), pBlueBac (Invitrogen), pBAcPAK (Clontech), pHIL (Invitrogen), pYES2 (Invitrogen), pCDNA (Invitrogen), pREP (Invitrogen) or the like.

The expression vector may be introduced into host cells via any one of a number of techinques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation.

E. coli containing an expression plasmid with the target gene fused to FKBP are grown and appropriately induced. The cells are then pelleted and resuspended in a suitable buffer. Although FKBP- 12 lacks sequences that specifically direct it to the periplasm, FKBP fusions are primarily located there and can be released by a standard freeze/thaw treatment of the cell pellet. Following centrifugation, the resulting supematant contains >80% pure FKBP fusion, which if desired can be purified further by conventional methods. Alternatively, the assay is not dependent on pure protein and the initial periplasmic preparation may be used directly. A thrombin site located between FKBP and the target protein can be used as a means to cleave FKBP from the fusion; such cleaved material may be a suitable negative control for subsequent assays.

A fusion protein which contains a single or multiple SH2 domain(s) may be purified by preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support. A freeze/thaw extract is prepared from the cells which express the fusion protein and is loaded onto the affinity matrix. The desired fusion protein is then specifically eluted with phenyl phosphate.

To assay the formation of a complex between a target protein and its ligand, the tagged ligand is mixed with the FKBP fusion protein in a suitable buffer in the presence of the radiolabeled ligand in the well of a white micropiate. After a suitable incubation period to allow complex formation to occur, coated SPA beads are added to capture the tagged ligand and any bound fusion protein. The plate is sealed, incubated for a sufficient period to allow the capture to go to completion, then counted in a multiwell scintillation counter. Screening for agonists/antagonists/inhibitors is carried out by performing the initial incubation prior to the capture step with SPA beads in the presence of a test compound(s) to determine whether they have an effect upon the binding of the tagged ligand to the fusion protein. This principle is illustrated by Figure 1.

The present invention can be understood further by the following examples, which do not constitute a limitation of the invention.

EXAMPLE 1

Process for Preparing the FKBP fusion cloning vector

General techniques for modifying and expressing genes in various host cells can be found in Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. Current Protocols in Molecular Biology (John Wiley & Sons, New York, New York, 1989). Sequences for a 3'- altered FKBP fragment that contained a glycine codon (GGT) in place of the stop (TGA) codon followed by a sequence encoding a thrombin site (Leu-Val-Pro-Arg) and BamHI restriction site (GAATTC) were amplified using the polymerase chain reaction (PCR). The PCR reaction contained the following primers:5'- GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' and 5'- TACGAATTCTGGCGTGGATCCACGCGGAACCAGACCTTCCAGT TTTAG-3' and a plasmid containing human FKBP- 12 as the template. The resulting 367 base pair amplification product was ligated into the vector pCRII (Invitrogen) and the ligation mixture transformed into competent Escherichia coli cells. Clones containing an insert were identified using PCR with flanking vector primers. Dideoxy DNA sequencing confirmed the nucleotide sequence of one positive isolate. The altered 338 base pair FKBP fragment was excised from the pCRII plasmid using Ncol and BamHI and ligated into Ncol anditømHI digested pET9d (Novagen) plasmid. Competent E. coli were transformed with the ligation mixture, and colonies containing the insert were identified using PCR with primers encoding for flanking vector sequences. The FKBP fusion cloning vector is called pET9dFKBPt. EXAMPLE 2

Process for Preparing the FK-ZAP fusion expression vector A DNA fragment encoding for the tandem SH2 domains of

ZAP-70 was prepared by PCR to contain a BamHI site at the 5'-end such that the reading frame was conserved with that of FKBP in the fusion vector. At the 3'-end, the fragment also incoφorated a stop codon followed by a BamHI site. The PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:

5 -ATTAGGATCCATGCCAGATCCTGCAGCTCACCTGCCCT-3 and S -ATATGGATCCTTACCAGAGGCGTTGCT-S'. The fragment was cloned into a suitable vector, sequenced, digested with BamHI, and the insert containing the SH2 domains ligated to BamHI treated pET9dFKBPt, and transformed into E. coli. Clones containing inserts in the correct orientation were identified by PCR or restriction enzyme analysis. Plasmid DNA was prepared and used to transform BL21 (DE3) cells.

EXAMPLE 3

Process for Preparing the FK-SYK fusion expression vector

The expression vector for the tandem SH2 domains of Syk fused to FKBP was prepared as in Example 2 except that the PCR reaction contained Raji cell cDNA (Clontech) and the following primers: 5 -CAATAGGATCCATGGCCAGCAGCGGCATGGCTGA-3 and 5 -GACCTAGGATCCCTAATTAACATTTCCCTGTGTGCCGAT-3^* .

EXAMPLE 4

Process for Preparing the FK-LCK fusion expression vector

The expression vector for the SH2 domain of Lck fused to FKBP was prepared as in Example 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers: 5 -ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3' and 5 -ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTGT

GCA-3" .

EXAMPLE 5

FK-ZAP

Step A: Process for Expression of FK-ZAP

E. coli BL21(DE3) cells containing the pET9dFKBPt/ ZapSH2 plasmid were grown in Luria-Bertani (LB) media containing 50 microgram/ml kanamycin at 37 degrees C until the optical density measured at 600 nm was 0.5-1.0. Expression of the FK-ZAP fusion protein was induced with 0.1 mM isopropyl beta-thiogalactopyranoside and the cells were grown for another 3-5 hr at 30 degrees C. They were pelleted at 4400 x g for 10 min at 4 degrees C and resuspended in 2% of the original culture volume with 100 mM tris pH 8.0 containing 1 microgram/ml each aprotinin, pepstatin, leupeptin, and bestatin. The resuspended pellet was frozen at -20 degrees C until further purification.

Step B: Process for Purification of FK-ZAP

The affinity matrix for purification of FK-ZAP was prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the ζl ITAM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK, and washing out unbound peptide. Frozen cells containing FK-ZAP were thawed in warm water, refrozen on dry ice for about 25 min., then thawed again. After the addition of 0.1 % octyl glucoside, 1 mM dithiothreitol (DTT) and 500 mM NaCl, the extract was centrifuged at 35,000 x g for approximately 30 minutes. The supernatant was loaded onto the phosphopeptide affinity column, at about 4° and washed with phosphate buffered saline containing 1 mM DTT and 0.1 % octyl glucoside. FK-ZAP was eluted with 200 mM phenyl phosphate in the same buffer at about 37°. The protein pool was concentrated and the phenyl phosphate removed on a desalting column. The purified FK-ZAP was stored at about -30° in 10 mM HEPES/150 mM NaCl/1 mM DTT/0.1 mM EDTA/10% glycerol.

EXAMPLE 6

FK-SYK

E. coli BL21(DE3) cells containing the pET9dFKBPt/ SykSH2 plasmid were grown, induced, and harvested as described in Example 5. FK-SYK was purified using the same affinity matrix and methodology described in Example 5.

EXAMPLE 7

FK-LCK

E. coli BL21(DE3) cells containing the pET9dFKBPt/ LckSH2 plasmid were grown, induced, and harvested as described in Example 5. The affinity matrix for purification of FK-LCK was prepared by combining agarose-immobilized avidin with excess biotinyl-

EPQpYEEIPIYL, and washing out unbound peptide. The remaining methodology for purification was the same as Example 5.

EXAMPLE 8

Method of Screening for Antagonists of FK-ZAP

Assays were conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01 % TWEEN-20, pH 7.0. 10 μl of a DMSO solution of test compound(s) and 120 μl of biotinyl- phosphopeptide stock solution were dispensed into the wells of a 96-well Packard Optiplate. Next, 20 μl of a mixture of FK-ZAP protein and 3H-dihydroFK506 were added to each test well. Finally, 50 μl of a 4 mg/ml suspension of SPA beads were dispensed to each well. Final concentrations of the assay components were: 25 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK 25 nM FK-ZAP fusion protein 10 nM 3H-dihydroFK506 (DuPont NEN) 1.0 mg/ml streptavidin-SPA beads (Amersham) 5% DMSO

The plate was sealed and incubated between 1 and 8 hours. Bead-bound radioactivity was then measured in a Packard Topcount micropiate scintillation counter.

EXAMPLE 9

Method of Screening for Antagonists of FK-SYK

The assays were conducted as set forth in Example 8, except that FK-SYK replaced FK-ZAP.

EXAMPLE 10

Method of Screening for Antagonists of FK-LCK

The assays were conducted as set forth in Example 8, except that FK-LCK replaced FK-ZAP and the tagged ligand was 25 nM biotiny 1 -EPQp YEEIPI YL.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Salowe, Scott P.

(ii) TITLE OF INVENTION: A High Throughput Assay Using Fusion Proteins

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Valerie J. Camara

(B) STREET: 126 E. Lincoln Avenue, P.O. Box 2000

(C) CITY: Rahway

(D) STATE: NJ

(E) COUNTRY: U.S.A.

(F) ZIP: 07065

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Camara, Valerie J.

(B) REGISTRATION NUMBER: 35,090

(C) REFERENCE/DOCKET NUMBER: 19494

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (908) 594-3902

(B) TELEFAX: (908) 594-4720

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1137 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60

CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 120

CGGGACAGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 180

GAAGAAGGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 2 0

TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTT_C 300

GATGTGGAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGCC AGATCCTGCA 360

GCTCACCTGC CCTTCTTCTA CGGCAGCATC TCGCGTGCCG AGGCCGAGGA GCACCTGAAG 420

CTGGCGGGCA TGGCGGACGG GCTCTTCCTG CTGCGCCAGT GCCTGCGCTC GCTGGGCGGC 480

TATGTGCTGT CGCTCGTGCA CGATGTGCGC TTCCACCACT TTCCCATCGA GCGCCAGCTC 5 0

AACGGCACCT ACGCCATTGC CGGCGGCAAA GCGCACTGTG GACCGGCAGA GCTCTGCGAG 600

TTCTACTCGC GCGACCCCGA CGGGCTGCCC TGCAACCTGC GCAAGCCGTG CAACCGGCCG 660

TCGGGCCTCG AGCCGCAGCC GGGGGTCTTC GACTGCCTGC GAGACGCCAT GGTGCGTGAC 720

TACGTGCGCC AGACGTGGAA GCTGGAGGGC GAGGCCCTGG AGCAGGCCAT CATCAGCCAG 780

GCCCCGCAGG TGGAGAAGCT CATTGCTACG ACGGCCCACG AGCGGATGCC CTGGTACCAC 840

AGCAGCCTGA CGCGTGAGGA GGCCGAGCGT AAACTTTACT CTGGGGCGCA GACCGACGGC 900

AAGTTCCTGC TGAGGCCGCG GAAGGAGCAG GGCACATACG CCCTGTCCCT CATCTATGGG 960

AAGACGGTGT ACCACTACCT CATCAGCCAA GACAAGGCGG GCAAGTACTG CATTCCCGAG 1020

GGCACCAAGT TTGACACGCT CTGGCAGCTG GTGGAGTATC TGAAGCTGAA GGCGGACGGG 1080

CTCATCTACT GCCTGAAGGA GGCCTGCCCC AACAGCAGTG CCAGCAACGC CTCTTAA 1137 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1155 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60

CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 120 CGGGACAGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 180

GAAGAAGGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240

TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTTC 300

GATGTGGAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGGC CAGCAGCGGC 360

ATGGCTGACA GCGCCAACCA CCTGCCCTTC TTTTTCGGCA ACATCACCCG GGAGGAGGCA 420

GAAGATTACC TGGTCCAGGG GGGCATGAGT GATGGGCTTT ATTTGCTGCG CCAGAGCCGC 480

AACTACCTGG GTGGCTTCGC CCTGTCCGTG GCCCACGGGA GGAAGGCACA CCACTACACC 540

ATCGAGCGGG AGCTGAATGG CACCTACGCC ATCGCCGGTG GCAGGACCCA TGCCAGCCCC 600

GCCGACCTCT GCCACTACCA CTCCCAGGAG TCTGATGGCC TGGTCTGCCT CCTCAAGAAG 660

CCCTTCAACC GGCCCCAAGG GGTGCAGCCC AAGACTGGGC CCTTTGAGGA TTTGAAGGAA 720

AACCTCATCA GGGAATATGT GAAGCAGACA TGGAACCTGC AGGGTCAGGC TCTGGAGCAG 780

GCCATCATCA GTCAGAAGCC TCAGCTGGAG AAGCTGATCG CTACCACAGC CCATGAAAAA 840

ATGCCTTGGT TCCATGGAAA AATCTCTCGG GAAGAATCTG AGCAAATTGT CCTGATAGGA 900

TCAAAGACAA ATGGAAAGTT CCTGATCCGA GCCAGAGACA ACAACGGCTC CTACGCCCTG 960

TGCCTGCTGC ACGAAGGGAA GGTGCTGCAC TATCGCATCG ACAAAGACAA GACAGGGAAG 1020

CTCTCCATCC CCGAGGGAAA GAAGTTCGAC ACGCTCTGGC AGCTAGTCGA GCATTATTCT 1080

TATAAAGCAG ATGGTTTGTT AAGAGTTCTT ACTGTCCCAT GTCAAAAAAT CGGCACACAG 1140

GGAAATGTTA ATTAG 1155 ( 2 ) INFORMATION FOR SEQ ID NO : 3 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 675 base pairs

(B) TYPE : nucleic acid

(C ) STRANDEDNESS : single

(D) TOPOLOGY : linear

( ii ) MOLECULE TYPE : DNA (genomic )

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 3 :

ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60

GAAGAAGGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240

TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTTC 300

GATGTGGAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGGC GAACAGCCTG 360

GAGCCCGAAC CCTGGTTCTT CAAGAACCTG AGCCGCAAGG ACGCGGAGCG GCAGCTCCTG 420

GCGCCCGGGA ACACTCACGG CTCCTTCCTC ATCCGGGAGA GCGAGAGCAC CGCGGGATCG 480

TTTTCACTGT CGGTCCGGGA CTTCGACCAG AACCAGGGAG AGGTGGTGAA ACATTACAAG 540

ATCCGTAATC TGGACAACGG TGGCTTCTAC ATCTCCCCTC GAATCACTTT TCCCGGCCTG 600

CATGAACTGG TCCGCCATTA CACCAATGCT TCAGATGGGC TGTGCACACG GTTGAGCCGC 660

CCCTGCCAGA CCTAA 675 (2) INFORMATION FOR SEQ ID Nθ:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 378 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Gly Val Gin Val Glu Thr lie Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15

Pro Lys Arg Gly Gin Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30

Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45

Phe Met Leu Gly Lys Gin Glu Val lie Arg Gly Trp Glu Glu Gly Val 50 55 60

Ala Gin Met Ser Val Gly Gin Arg Ala Lys Leu Thr lie Ser Pro Asp 65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly lie lie Pro Pro His Ala 85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro 100 105 110 Arg Gly Ser Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly 115 120 125

Ser lie Ser Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met 130 135 140

Ala Asp Gly Leu Phe Leu Leu Arg Gin Cys Leu Arg Ser Leu Gly Gly 145 150 155 160

Tyr Val Leu Ser Leu Val His Asp Val Arg Phe His His Phe Pro lie 165 170 175

Glu Arg Gin Leu Asn Gly Thr Tyr Ala lie Ala Gly Gly Lys Ala His 180 185 190

Cys Gly Pro Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly 195 200 205

Leu Pro Cys Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu 210 215 220

Pro Gin Pro Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp 225 230 235 240

Tyr Val Arg Gin Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gin Ala 245 250 255

He He Ser Gin Ala Pro Gin Val Glu Lys Leu He Ala Thr Thr Ala 260 265 270

His Glu Arg Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala 275 280 285

Glu Arg Lys Leu Tyr Ser Gly Ala Gin Thr Asp Gly Lys Phe Leu Leu 290 295 300

Arg Pro Arg Lys Glu Gin Gly Thr Tyr Ala Leu Ser Leu He Tyr Gly 305 310 315 320

Lys Thr Val Tyr His Tyr Leu He Ser Gin Asp Lys Ala Gly Lys Tyr 325 330 335

Cys He Pro Glu Gly Thr Lys Phe Asp Thr Leu Trp Gin Leu Val Glu 340 345 350

Tyr Leu Lys Leu Lys Ala Asp Gly Leu He Tyr Cys Leu Lys Glu Ala 355 360 365

Cys Pro Asn Ser Ser Ala Ser Asn Ala Ser 370 375 ) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 384 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Gly Val Gin Val Glu Thr He Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15

Pro Lys Arg Gly Gin Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30

Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45

Phe Met Leu Gly Lys Gin Glu Val He Arg Gly Trp Glu Glu Gly Val 50 55 60

Ala Gin Met Ser Val Gly Gin Arg Ala Lys Leu Thr He Ser Pro Asp 65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly He He Pro Pro His Ala 85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro 100 105 110

Arg Gly Ser Met Ala Ser Ser Gly Met Ala Asp Ser Ala Asn His Leu 115 120 125

Pro Phe Phe Phe Gly Asn He Thr Arg Glu Glu Ala Glu Asp Tyr Leu 130 135 140

Val Gin Gly Gly Met Ser Asp Gly Leu Tyr Leu Leu Arg Gin Ser Arg 145 150 155 160

Asn Tyr Leu Gly Gly Phe Ala Leu Ser Val Ala His Gly Arg Lys Ala 165 170 175

His His Tyr Thr He Glu Arg Glu Leu Asn Gly Thr Tyr Ala He Ala 180 185 190

Gly Gly Arg Thr His Ala Ser Pro Ala Asp Leu Cys His Tyr His Ser 195 200 205

Gin Glu Ser Asp Gly Leu Val Cys Leu Leu Lys Lys Pro Phe Asn Arg 210 215 220

Pro Gin Gly Val Gin Pro Lys Thr Gly Pro Phe Glu Asp Leu Lys Glu 225 230 235 240 Asn Leu He Arg Glu Tyr Val Lys Gin Thr Trp Asn Leu Gin Gly Gin 245 250 255

Ala Leu Glu Gin Ala He He Ser Gin Lys Pro Gin Leu Glu Lys Leu 260 265 270

He Ala Thr Thr Ala His Glu Lys Met Pro Trp Phe His Gly Lys He 275 280 285

Ser Arg Glu Glu Ser Glu Gin He Val Leu He Gly Ser Lys Thr Asn 290 295 300

Gly Lys Phe Leu He Arg Ala Arg Asp Asn Asn Gly Ser Tyr Ala Leu 305 310 315 320

Cys Leu Leu His Glu Gly Lys Val Leu His Tyr Arg He Asp Lys Asp 325 330 335

Lys Thr Gly Lys Leu Ser He Pro Glu Gly Lys Lys Phe Asp Thr Leu 340 345 350

Trp Gin Leu Val Glu His Tyr Ser Tyr Lys Ala Asp Gly Leu Leu Arg 355 360 365

Val Leu Thr Val Pro Cys Gin Lys He Gly Thr Gin Gly Asn Val Asn 370 375 380

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 224 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Gly Val Gin Val Glu Thr He Ser Pro Gly Asp Gly Arg Thr Phe

1 5 10 15

Pro Lys Arg Gly Gin Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30

Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45

Phe Met Leu Gly Lys Gin Glu Val He Arg Gly Trp Glu Glu Gly Val 50 55 60 Ala Gin Met Ser Val Gly Gin Arg Ala Lys Leu Thr He Ser Pro Asp 65 70 75 80

Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly He He Pro Pro His Ala 85 90 95

Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro 100 105 no

Arg Gly Ser Met Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe Phe Lys 115 120 125

Asn Leu Ser Arg Lys Asp Ala Glu Arg Gin Leu Leu Ala Pro Gly Asn 130 135 140

Thr His Gly Ser Phe Leu He Arg Glu Ser Glu Ser Thr Ala Gly Ser 145 150 155 160

Phe Ser Leu Ser Val Arg Asp Phe Asp Gin Asn Gin Gly Glu Val Val 165 170 175

Lys His Tyr Lys He Arg Asn Leu Asp Asn Gly Gly Phe Tyr He Ser 180 185 190

Pro Arg He Thr Phe Pro Gly Leu His Glu Leu Val Arg His Tyr Thr 195 200 205

Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys Gin Thr 210 215 220

Claims

WHAT IS CLAIMED IS:

1. A method of screening for compounds capable of binding to a fusion protein which comprises the steps of: a) mixing a test compound, a tagged ligand, the fusion protein, a radiolabeled ligand and coated scintillation proximity assay (SPA) beads; b) incubating the mixture from between about 1 hour to about 24 hours; c) measuring the SPA bead-bound counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound using scintillation counting; and d) determining the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a control assay run in the absence of the test compound.

2. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 1 , wherein the tagged ligand is a biotinylated ligand or epitope-tagged ligand.

3. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 2, wherein scintillation proximity assay beads are streptavidin-coated or anti-antibody or protein A-coated.

4. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 3, wherein the radiolabeled ligand consists of [^H]- or [125τ]_ι_ab_eι_ed FK506 analog.

5. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 4, wherein the fusion protein comprises an FK506-binding protein linked through a peptide linker to a target protein.

6. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 5, wherein the target protein comprises a single or multiple signal transduction domain.

7. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 6, wherein the single or multiple signal transduction domain is selected from the group consisting of: SHI , SH2, SH3 and PH domains.

8. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 7, wherein the target protein is a single or multiple SH2 domain.

9. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 8, wherein the radiolabeled ligand is [3H]-dihydroFK506.

10. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 9, wherein the FK506- binding protein is a 12kDA human FK506-binding protein.

1 1. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 10, wherein the target protein is a single or multiple SH2 domain selected from the group consisting of: ZAP:SH2, SYK:SH2 and LCK:SH2.

12. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 1 1 , wherein the target protein is the SH2 domain, ZAP:SH2.

13. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 11 , wherein the target protein is the SH2 domain, SYK:SH2.

14. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 11 , wherein the target protein is the SH2 domain, LCK:SH2.