CA2231385A1 - A high throughput assay using fusion proteins - Google Patents
A high throughput assay using fusion proteins Download PDFInfo
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- CA2231385A1 CA2231385A1 CA002231385A CA2231385A CA2231385A1 CA 2231385 A1 CA2231385 A1 CA 2231385A1 CA 002231385 A CA002231385 A CA 002231385A CA 2231385 A CA2231385 A CA 2231385A CA 2231385 A1 CA2231385 A1 CA 2231385A1
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- binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9493—Immunosupressants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
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
;949~ P ~ ~ ~ 6 ~ 1 4 IP~VIIS 1 6 SEP 19~7 TITLE OF THE INVENTION
A HIGH THROUGHPUT ASSAY USING F~JSION PROTEINS
BA~KGRQUND OF THE INVENTION
Src homology 2 (SH2) domains are a family of homologous protein domains that share the cornmon property of recogni7in~;
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 straighlrol ~al-l af~mity purification on glutathione-Sepharose. Ligand binding is then assayed by incubating the GST/SH2 with a radiolabeled phosphopeptide, preci~ g the complex with glutathione-Sepharose, washing the beads, and then counting the beads to detçrrnine 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 t32P]ATP or chemically with [125I]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 applvpliate kinase must also be available in sufficient quantity to generate enough m~teri~l 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 ~rote,lls and ligands that have been applied to SH2 domains are biospecific interaction analysis using surface plasmon resonance and ~S~
~ CA 0223138~ 1998-03-06 9 ~ ~ 1 4 ~ 6 3 949~ ;16SEPl997 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); Lemmon 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.
,r~., 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 scintill~tion proximity assay (SPA) beads, and then measuring the scintill~tion 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 m~kin~ 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 speci~li7er1 radiochemical synth~si.c and is readily adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors.
~r ~ CA 02231385 1998-03-06 - pCTI~;9~14563 19491 I~S 1 6 SEP 1~97 BRIEF DESCRlPTION 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 .
DF.TAILED DESCRIPTION OF THE INVENTION
The 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 scintill~tion 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 scintill~tion counting;
and d) determinin~ the binding of the tagged ligand to the fusion yrotein 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 "FK~06-binding protein" (FKBP), the two yroteills being separated by a "peptide linker".
A "peptide linker" may consist of a sequence cont~inin~
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 GLVPRGS (SEQ. ID. NO. 7).
A~S~r S;96 '1456}
i949~ ~ ~ 16 S E ~ t3~7 The terrn "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 limLited to, Src homology 1 (SHl), 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., 7, 306-311 (1995); Kuriyan &
Cowburn, Curr. Opin. Struct. Biol., 3, 828-837 (1993); Cohen et al., Cell, 80, 237-248 (1995)]. The term "SH1 domain" refers to a farnily 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 recogni7in~ phosphorylated tyrosine residues in specific peptide contexts. The term "SH3 domain" refers to a family of homologous protein domains that share the common ~lo~elLy of recogni7in~ 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 lltili7e~1 in the method of the invention include, but are not limite~
to, the single and tandem SH2 domains present in the tyrosine kin~es 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: hllm~n ZAP (L05 148); hllm~n SYK (L28824) and human LCK (X13529). The sequences for ZAP, Syk and Lck are disclosed in the sequence listing as follows: the isolated DNA encoding for a fusion ~>loteill cont~inin~ ~AP iS (SEQ.ID.NO.l);
the isolated DNA encoding for a fusion protein cont~inin~ Syk is (SEQ.ID.NO.2); the isolated DNA encoding for a fusion protein cont~inin~ Lck is (SEQ.ID.NO.3); the sequence for the FKBP-ZAP:SH2 fusion protein is (SEQ.ID.NO.4); the sequence for the FKBP-Syk:SH2 fusion protein is (SEQ.ID.NO.5); and the sequence for the F~BP-Lck:SH2 fusion protein is (SEQ.ID.NO.6).
The term "tagged ligand" refers to a biotinylated or epitope tagged ligand for the target protein.
~ e.$v~
. CA 02231385 1998-03-06 ~ ~: J 3 l i"7~ ' 6 4g~ ~ . L "" ,3 jr~
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 streptavidin-coated scintil1~tion proximity assay beads when the tagged ligand is biotinylated, and to anti-epitope antibody bound to anti-antibody-coated or protein A-coated scintill~tion 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 scintill~tion proximity assay beads, but in the absence of the test compound.
The term F'K506-binding ~l~otei~s may include, but are not limit~-l 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 ~e scope of the invention.
M~mm~liqn FKBP-12 Galat et al., Eur. J. Biochem., 216:689-707(1993).
~ FKBP-12.6 Wiede~echt, G. and F. Et_ko~
Perspectives in Drug Discovery and Design, 2:57-84(1994).
FKBp-l3 Galat et al., supra; Wiederrecht and Etzkom, supra.
FKBP-25 Galat et al., supra; Wiederrecht and Ft7.ko~, supra.
FKBP-38 Wiederrecht and Etzkorn, supra.
FKBP-Sl B~ hm~n et al., Mol. Cell. Biol., 8, 4395-4402(1995).
FKBP-52 Galat et al., supra.
P~; 9 ~ ~ I 4 ~6 '1949~ ~ ~ 16 SEP 1997 l~acteria Legionella pneumophilia Galat et al., supra.
Legionella micadei Galat et al., supra.
Chlamydia trachomatis Galat et al., supra.
E. coli fkpa Horne, 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 Tr~n-linh 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.
Fnn~
yeast FKBP-12 Cardenas et al., Perspectives in Drug Discovery and Design, 2: 103-126 (1994).
yeast FKBP-13 Cardenas et al., supra.
yeast NPRl(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 limite-l to, bacteria, yeast, bluegreen algae, plant cells, insect cells and ~nim~l cells.
Expression vectors are defin~ l herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an ~ rop.iate 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 ~nim~l cells.
Specifically designed vectors allow the ~hllttlin~ of DNA
between hosts such as bacteria-yeast or bacteria-~nim~l cells. An a~ro~,liately constructed expression vector may contain: an origin of ~ J ~J .'~ ; 6 3 19491 ~$ ~ fi SLP 1~97 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 pl~mi-~s or viruses. Commercially available vectors suitable for FKBP
fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pl 7 (USB), pET (Novagen), pIBI (IBI), pProEX-l (Gibco/BRL), pBluescript II (Stratagene), plZ18R 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 limite-l to transformation, transfection, infection, protoplast fusion, and electroporation .
E. coli cont~inin~: an expression plasmid with the target gene e-S~:~ fused to FKBP are grown and a~ro~liately in~ cecl 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 SUp~ t contains >80% pure FKBP fusion, which if desired can be purified fur~er 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 m~tç~ l 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 -~ r !
949~ ~ S 1 6 irnmobilized on a solid support~ A freeze/thaw extract is prepared from the cells which express the fusion protein and is loaded onto the af~mity matrix. The desired filsion 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 rnixed with the FKBP fusion protein in a suitable buffer in the presence of the radiolabeled ligand in the well of a white microplate. 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 suf~lcient period to allow the capture to go to completion, then counted in a multiwell scintill~tion 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.
Ihe present invention can be understood ~urther by the following examples, which do not constitute a limitation of the invention.
., F.XAMPr,F. 1 Process for Preparin~ the FKBP fusion clonin~ 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., Se~ n~ 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 E~KBP 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:S'-GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' (SEQ.ID.NO.8) and S'-TACGAA'~CTGGCGTGGATCCACGCGG
- P~TIU~9~4~63 1~491 ~ C P 19~7 AACCAGACCTTCCA~ l l l-l AG-3' (SEQ.ID.NO.9) and a plasrnid con~inin~ 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 cont~inin~ an insert were identified using PCR with tl~nkin~
vector primers. Dideoxy DNA sequencing conf~ed the nucleotide sequence of one positive isolate. The altered 338 base pair FKBP
fragment was excised from the pCRII plasmid using NcoI and BamHI
and ligated into NcoI andBamHI digested pET9d (Novagen) plasmid.
Competent E. coli were transforrned with the ligation mixture, and colonies cont~inin~ the insert were identified using PCR with primers encoding for fl~nking vector sequences. The FKBP fusion cloning vector is called pET9dFKBPt.
Process for Preparinp the FK-ZAP fusion expression vector A DNA fragment encoding for the t~n~çm 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 incorporated a stop codon followed by a BamHI site. The PCR reaction contained Molt-4 cDNA
(Clontech) and the following primers:
S'-ATTAGGATCCATGCCAGATCCTGCAGCTCACCTGCCCT-3' (SEQ.ID.NO.lO) and S'-ATATGGATCCTTACCAGAGGCGTTGCT-3' (SEQ.ID.NO. l l). The fragment was cloned into a suitable vector, sequenced, digested with BamXI, and the insert cont~inin~ the SH2 domains ligated to BamHI treated pET9dFKBPt, and transformed into E.
coli. Clones con~inin~ inserts in the correct orientation were identified by PCR or restriction enzyme analysis. Plasmid DNA was prepared and used to transforrn BL21(DE3) cells.
AN~
~ CA 0223138~ 1998-03-06 q~ ~4~
1 949~ 7 EXAMPI.E 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 ' (SEQ.ID.NO. 12) and S'-GACCTAGGATCCCTAATTAACATl~CCC
TGTGTGCCGAT-3' (SEQ.ID.NO. 13).
FXAMPT,F. 4 Process for Preparin,~ the FK-LCK fusion expression vector The expression vector for the SH2 domain of Lck fused to FKBP was prepared as in F.~mple 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
5 '-ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3 ' (SEQ.ID.NO. 14) and 5'-ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTGT
GCA-3' (SEQ.ID.NO. 15).
f''~
F,XAMPLE S
-FK-7:AP
Step ~: Process for F,xpression of FK-ZAP
E. coli BL21(DE3) cells cont~ining the pET9dFKBPt/
ZapSH2 plasmid were grown in Luria-Bertani (LB) media cont~inin?~ 50 microgram/ml k~n~mycin at 37 degrees C until the optical density measured at 600 nm was 0.5-1Ø Expression of the F~-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 cont~inin~ 1 - -, CA 0223138S 1998-03-06 '1949~ ~ 9~ /~4~63 ~P~WS 1~ SEP 1997 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-Z:AP was prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the ~1 ITAM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK (SEQ.ID.NO.
r~-. 16), and washing out unbound peptide. Frozen cells cont~inin~ 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 approxim~tely 30 rninutes. The supern~t~nt was loaded onto the phosphopeptide affinity column, at about 4~ and washed with phosphate buffered saline cont~inin~ 1 mM Dll and 0.1% octyl glucoside. FK-ZAP was eluted wi~ 200 mM phenyl phosphate in the same buffer at about 37~. The protein pool was concentrated and the phenyl phosphate removed on a ~es~l*n~ column. The purified FK-ZAP
was stored at about -30~ in 10 mM HEPES/150 rnM NaCl/l mM
{ DTT/0. 1 rnM EDTA/10% glycerol.
FK-~YK
E. coli BL21(DE3) cells co..l~ i..g ~e pET9dFKBPt/
- SykSH2 pl~micl 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.
FxAMpLE 7 FK-LCK
E. coli BL21(DE3) cells cont~ining the pET9dFKBPtl LckSH2 plasmid were grown, induced, and harvested as described in P~Q~DS~
~ CA 02231385 1998-03-06 ~7~9~1145~o~
19491 ~ P 1~37 Example 5. The affinity matrix for purification of FK-LCK was prepared by combining agarose-immobilized avidin with excess biotinyl-EPQpYEEIPIYL (SEQ.ID.NO. 17), and washing out unbound peptide.
The rem~inin~ methodology for purification was the same as Example 5.
Method of Screenin~ for Ant~onists of FK-ZAP
Assays were conducted at ambient ten~e.~lule in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01% TWEEN-20, pH 7Ø
10 ,ul of a DMSO solution of test compound(s) and 120 ~1 of biotinyl-phosphopeptide stock solution were dispensed into the wells of a 96-well Packard Optiplate. Next, 20 111 of a mixture of FK-ZAP protein arld 3H-dihydroFK506 were added to each test well. Finally, 50 ,ul of a 4 mglml suspension of SPA beads were dispensed to each well. Final concentrations of the assay components were:
25 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK
(SEQ.ID.NO. 16) 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 microplate scintill~tion counter.
Method of Screenin.~ for Anta~onists of FK-SYK
The assays were conducted as set forth in Exarnple 8, except that FK-SYK replaced FK-ZAP.
~r~
. CA 02231385 1998-03-06 j96~14563 19491 ~ S 1 6SEP19~7 E~MPLE 10 Method of Screening for ~nt~onists of FE~-LCK
The assays were conducted as set for~ in Example 8, except that FK-LCK replaced FK-ZAP and the tagged ligand was 25 nM
biotinyl-EPQpYEEIPIYL (SEQ.ID.NO. 17).
,~ .
..
~ CA 0223l385 l998-03-06 19494 ~ t! ;~ F~ ~ t9~
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: SALOWE, SCOTT P.
(ii) TITLE OF THE INVENTION: A HIGH THROu~ul ASSAY USING
FUSION PROTEINS
(iii) NUMBER OF SEQUENCES: 17 (iv) CORRESP~N~ ADDRESS:
~ (A) ADDRESSEE: Merck & Co., Inc.
~ (B) STREET: P.O. Box 2000, 126 E. Lincoln Ave.
(C) CITY: Rahway (D) STATE: NJ
(E) COUNTRY: USA
(F) ZIP: 07065-0900 (v) COMPUTER READA8LE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/707,793 (B) FILING DATE: 04-SEP-1996 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) AllOnN~Y/AGENT INFORMATION:
(A) NAME: Camara, Valerie J
(8) REGISTRATION NUMBER: 35,090 (C) REFERENCE/DOCKET NUM8ER: 19494 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-3902 (B) TELEFAX: 908-594-4720 (C) TELEX:
~E~ED S~T
~ CA 0223l38S l998-03-06 ~19494 ~ C ~ ~ ~ 9 6 / 1 4 5 6 ~
A HIGH THROUGHPUT ASSAY USING F~JSION PROTEINS
BA~KGRQUND OF THE INVENTION
Src homology 2 (SH2) domains are a family of homologous protein domains that share the cornmon property of recogni7in~;
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 straighlrol ~al-l af~mity purification on glutathione-Sepharose. Ligand binding is then assayed by incubating the GST/SH2 with a radiolabeled phosphopeptide, preci~ g the complex with glutathione-Sepharose, washing the beads, and then counting the beads to detçrrnine 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 t32P]ATP or chemically with [125I]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 applvpliate kinase must also be available in sufficient quantity to generate enough m~teri~l 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 ~rote,lls and ligands that have been applied to SH2 domains are biospecific interaction analysis using surface plasmon resonance and ~S~
~ CA 0223138~ 1998-03-06 9 ~ ~ 1 4 ~ 6 3 949~ ;16SEPl997 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); Lemmon 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.
,r~., 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 scintill~tion proximity assay (SPA) beads, and then measuring the scintill~tion 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 m~kin~ 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 speci~li7er1 radiochemical synth~si.c and is readily adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors.
~r ~ CA 02231385 1998-03-06 - pCTI~;9~14563 19491 I~S 1 6 SEP 1~97 BRIEF DESCRlPTION 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 .
DF.TAILED DESCRIPTION OF THE INVENTION
The 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 scintill~tion 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 scintill~tion counting;
and d) determinin~ the binding of the tagged ligand to the fusion yrotein 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 "FK~06-binding protein" (FKBP), the two yroteills being separated by a "peptide linker".
A "peptide linker" may consist of a sequence cont~inin~
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 GLVPRGS (SEQ. ID. NO. 7).
A~S~r S;96 '1456}
i949~ ~ ~ 16 S E ~ t3~7 The terrn "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 limLited to, Src homology 1 (SHl), 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., 7, 306-311 (1995); Kuriyan &
Cowburn, Curr. Opin. Struct. Biol., 3, 828-837 (1993); Cohen et al., Cell, 80, 237-248 (1995)]. The term "SH1 domain" refers to a farnily 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 recogni7in~ phosphorylated tyrosine residues in specific peptide contexts. The term "SH3 domain" refers to a family of homologous protein domains that share the common ~lo~elLy of recogni7in~ 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 lltili7e~1 in the method of the invention include, but are not limite~
to, the single and tandem SH2 domains present in the tyrosine kin~es 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: hllm~n ZAP (L05 148); hllm~n SYK (L28824) and human LCK (X13529). The sequences for ZAP, Syk and Lck are disclosed in the sequence listing as follows: the isolated DNA encoding for a fusion ~>loteill cont~inin~ ~AP iS (SEQ.ID.NO.l);
the isolated DNA encoding for a fusion protein cont~inin~ Syk is (SEQ.ID.NO.2); the isolated DNA encoding for a fusion protein cont~inin~ Lck is (SEQ.ID.NO.3); the sequence for the FKBP-ZAP:SH2 fusion protein is (SEQ.ID.NO.4); the sequence for the FKBP-Syk:SH2 fusion protein is (SEQ.ID.NO.5); and the sequence for the F~BP-Lck:SH2 fusion protein is (SEQ.ID.NO.6).
The term "tagged ligand" refers to a biotinylated or epitope tagged ligand for the target protein.
~ e.$v~
. CA 02231385 1998-03-06 ~ ~: J 3 l i"7~ ' 6 4g~ ~ . L "" ,3 jr~
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 streptavidin-coated scintil1~tion proximity assay beads when the tagged ligand is biotinylated, and to anti-epitope antibody bound to anti-antibody-coated or protein A-coated scintill~tion 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 scintill~tion proximity assay beads, but in the absence of the test compound.
The term F'K506-binding ~l~otei~s may include, but are not limit~-l 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 ~e scope of the invention.
M~mm~liqn FKBP-12 Galat et al., Eur. J. Biochem., 216:689-707(1993).
~ FKBP-12.6 Wiede~echt, G. and F. Et_ko~
Perspectives in Drug Discovery and Design, 2:57-84(1994).
FKBp-l3 Galat et al., supra; Wiederrecht and Etzkom, supra.
FKBP-25 Galat et al., supra; Wiederrecht and Ft7.ko~, supra.
FKBP-38 Wiederrecht and Etzkorn, supra.
FKBP-Sl B~ hm~n et al., Mol. Cell. Biol., 8, 4395-4402(1995).
FKBP-52 Galat et al., supra.
P~; 9 ~ ~ I 4 ~6 '1949~ ~ ~ 16 SEP 1997 l~acteria Legionella pneumophilia Galat et al., supra.
Legionella micadei Galat et al., supra.
Chlamydia trachomatis Galat et al., supra.
E. coli fkpa Horne, 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 Tr~n-linh 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.
Fnn~
yeast FKBP-12 Cardenas et al., Perspectives in Drug Discovery and Design, 2: 103-126 (1994).
yeast FKBP-13 Cardenas et al., supra.
yeast NPRl(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 limite-l to, bacteria, yeast, bluegreen algae, plant cells, insect cells and ~nim~l cells.
Expression vectors are defin~ l herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an ~ rop.iate 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 ~nim~l cells.
Specifically designed vectors allow the ~hllttlin~ of DNA
between hosts such as bacteria-yeast or bacteria-~nim~l cells. An a~ro~,liately constructed expression vector may contain: an origin of ~ J ~J .'~ ; 6 3 19491 ~$ ~ fi SLP 1~97 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 pl~mi-~s or viruses. Commercially available vectors suitable for FKBP
fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pl 7 (USB), pET (Novagen), pIBI (IBI), pProEX-l (Gibco/BRL), pBluescript II (Stratagene), plZ18R 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 limite-l to transformation, transfection, infection, protoplast fusion, and electroporation .
E. coli cont~inin~: an expression plasmid with the target gene e-S~:~ fused to FKBP are grown and a~ro~liately in~ cecl 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 SUp~ t contains >80% pure FKBP fusion, which if desired can be purified fur~er 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 m~tç~ l 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 -~ r !
949~ ~ S 1 6 irnmobilized on a solid support~ A freeze/thaw extract is prepared from the cells which express the fusion protein and is loaded onto the af~mity matrix. The desired filsion 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 rnixed with the FKBP fusion protein in a suitable buffer in the presence of the radiolabeled ligand in the well of a white microplate. 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 suf~lcient period to allow the capture to go to completion, then counted in a multiwell scintill~tion 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.
Ihe present invention can be understood ~urther by the following examples, which do not constitute a limitation of the invention.
., F.XAMPr,F. 1 Process for Preparin~ the FKBP fusion clonin~ 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., Se~ n~ 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 E~KBP 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:S'-GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' (SEQ.ID.NO.8) and S'-TACGAA'~CTGGCGTGGATCCACGCGG
- P~TIU~9~4~63 1~491 ~ C P 19~7 AACCAGACCTTCCA~ l l l-l AG-3' (SEQ.ID.NO.9) and a plasrnid con~inin~ 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 cont~inin~ an insert were identified using PCR with tl~nkin~
vector primers. Dideoxy DNA sequencing conf~ed the nucleotide sequence of one positive isolate. The altered 338 base pair FKBP
fragment was excised from the pCRII plasmid using NcoI and BamHI
and ligated into NcoI andBamHI digested pET9d (Novagen) plasmid.
Competent E. coli were transforrned with the ligation mixture, and colonies cont~inin~ the insert were identified using PCR with primers encoding for fl~nking vector sequences. The FKBP fusion cloning vector is called pET9dFKBPt.
Process for Preparinp the FK-ZAP fusion expression vector A DNA fragment encoding for the t~n~çm 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 incorporated a stop codon followed by a BamHI site. The PCR reaction contained Molt-4 cDNA
(Clontech) and the following primers:
S'-ATTAGGATCCATGCCAGATCCTGCAGCTCACCTGCCCT-3' (SEQ.ID.NO.lO) and S'-ATATGGATCCTTACCAGAGGCGTTGCT-3' (SEQ.ID.NO. l l). The fragment was cloned into a suitable vector, sequenced, digested with BamXI, and the insert cont~inin~ the SH2 domains ligated to BamHI treated pET9dFKBPt, and transformed into E.
coli. Clones con~inin~ inserts in the correct orientation were identified by PCR or restriction enzyme analysis. Plasmid DNA was prepared and used to transforrn BL21(DE3) cells.
AN~
~ CA 0223138~ 1998-03-06 q~ ~4~
1 949~ 7 EXAMPI.E 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 ' (SEQ.ID.NO. 12) and S'-GACCTAGGATCCCTAATTAACATl~CCC
TGTGTGCCGAT-3' (SEQ.ID.NO. 13).
FXAMPT,F. 4 Process for Preparin,~ the FK-LCK fusion expression vector The expression vector for the SH2 domain of Lck fused to FKBP was prepared as in F.~mple 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
5 '-ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3 ' (SEQ.ID.NO. 14) and 5'-ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTGT
GCA-3' (SEQ.ID.NO. 15).
f''~
F,XAMPLE S
-FK-7:AP
Step ~: Process for F,xpression of FK-ZAP
E. coli BL21(DE3) cells cont~ining the pET9dFKBPt/
ZapSH2 plasmid were grown in Luria-Bertani (LB) media cont~inin?~ 50 microgram/ml k~n~mycin at 37 degrees C until the optical density measured at 600 nm was 0.5-1Ø Expression of the F~-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 cont~inin~ 1 - -, CA 0223138S 1998-03-06 '1949~ ~ 9~ /~4~63 ~P~WS 1~ SEP 1997 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-Z:AP was prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the ~1 ITAM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK (SEQ.ID.NO.
r~-. 16), and washing out unbound peptide. Frozen cells cont~inin~ 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 approxim~tely 30 rninutes. The supern~t~nt was loaded onto the phosphopeptide affinity column, at about 4~ and washed with phosphate buffered saline cont~inin~ 1 mM Dll and 0.1% octyl glucoside. FK-ZAP was eluted wi~ 200 mM phenyl phosphate in the same buffer at about 37~. The protein pool was concentrated and the phenyl phosphate removed on a ~es~l*n~ column. The purified FK-ZAP
was stored at about -30~ in 10 mM HEPES/150 rnM NaCl/l mM
{ DTT/0. 1 rnM EDTA/10% glycerol.
FK-~YK
E. coli BL21(DE3) cells co..l~ i..g ~e pET9dFKBPt/
- SykSH2 pl~micl 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.
FxAMpLE 7 FK-LCK
E. coli BL21(DE3) cells cont~ining the pET9dFKBPtl LckSH2 plasmid were grown, induced, and harvested as described in P~Q~DS~
~ CA 02231385 1998-03-06 ~7~9~1145~o~
19491 ~ P 1~37 Example 5. The affinity matrix for purification of FK-LCK was prepared by combining agarose-immobilized avidin with excess biotinyl-EPQpYEEIPIYL (SEQ.ID.NO. 17), and washing out unbound peptide.
The rem~inin~ methodology for purification was the same as Example 5.
Method of Screenin~ for Ant~onists of FK-ZAP
Assays were conducted at ambient ten~e.~lule in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01% TWEEN-20, pH 7Ø
10 ,ul of a DMSO solution of test compound(s) and 120 ~1 of biotinyl-phosphopeptide stock solution were dispensed into the wells of a 96-well Packard Optiplate. Next, 20 111 of a mixture of FK-ZAP protein arld 3H-dihydroFK506 were added to each test well. Finally, 50 ,ul of a 4 mglml suspension of SPA beads were dispensed to each well. Final concentrations of the assay components were:
25 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK
(SEQ.ID.NO. 16) 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 microplate scintill~tion counter.
Method of Screenin.~ for Anta~onists of FK-SYK
The assays were conducted as set forth in Exarnple 8, except that FK-SYK replaced FK-ZAP.
~r~
. CA 02231385 1998-03-06 j96~14563 19491 ~ S 1 6SEP19~7 E~MPLE 10 Method of Screening for ~nt~onists of FE~-LCK
The assays were conducted as set for~ in Example 8, except that FK-LCK replaced FK-ZAP and the tagged ligand was 25 nM
biotinyl-EPQpYEEIPIYL (SEQ.ID.NO. 17).
,~ .
..
~ CA 0223l385 l998-03-06 19494 ~ t! ;~ F~ ~ t9~
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: SALOWE, SCOTT P.
(ii) TITLE OF THE INVENTION: A HIGH THROu~ul ASSAY USING
FUSION PROTEINS
(iii) NUMBER OF SEQUENCES: 17 (iv) CORRESP~N~ ADDRESS:
~ (A) ADDRESSEE: Merck & Co., Inc.
~ (B) STREET: P.O. Box 2000, 126 E. Lincoln Ave.
(C) CITY: Rahway (D) STATE: NJ
(E) COUNTRY: USA
(F) ZIP: 07065-0900 (v) COMPUTER READA8LE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/707,793 (B) FILING DATE: 04-SEP-1996 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) AllOnN~Y/AGENT INFORMATION:
(A) NAME: Camara, Valerie J
(8) REGISTRATION NUMBER: 35,090 (C) REFERENCE/DOCKET NUM8ER: 19494 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-3902 (B) TELEFAX: 908-594-4720 (C) TELEX:
~E~ED S~T
~ CA 0223l38S l998-03-06 ~19494 ~ C ~ ~ ~ 9 6 / 1 4 5 6 ~
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1137 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
? GAAGAAGGGG TTGCCCAGAT GA~l~l~G~l CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240 TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC ~ C~T~llc 300 GCTCACCTGC C~ll~ll'~lA CGGCAGCATC TCGCGTGCCG AGGCCGAGGA GCACCTGAAG 420 AA~llC~l~C TGAGGCCGCG GAAGGAGCAG GGCACATACG CC~l~lCCCT CATCTATGGG 960 '- (2) INFORMATION FOR SEQ ID No:2:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 1155 base pairs (B) TYPE: nucleic acid (C) STRANI~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~u~ DESCRIPTION: SEQ ID NO:2:
ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCAC~llCCC CAAGCGCGGC 60 TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC ~ C~l~llC 300 ~ CA 0223l38~ l998-03-06 PC~ q~l456~
IP~WS16SEP1997 TCAAAGACAA ATGGAAAGTT CCTGATCCGA GCCA~A~-A~A ACAACGGCTC CTACGCCCTG 960 TATAAAGCAG A~ AAGAGTTCTT ACTGTCCCAT GTCAAAAAAT CGGCACACAG 1140 (2) INFORMATION FOR SEQ ID NO:3:
(i) ~QU~Nt_~ CHARACTERISTICS:
(A) LENGTH: 675 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAA~AAA~T TGATTCCTCC 120 TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC ~l~t_l~C~lt_llC 300 GAGCCCGAAC C~ CAAGAACCTG AGCCGCAAGG ACGCGGAGCG GCAGCTCCTG 420 GCGC'CCG~A ACACTCACGG ~lC~llCCTC ATCCGGGAGA GC~A~A~CAC CGCGGGATCG 480 ATCCGTAATC TG~A~AACGG TGG~ll~lAC ATCTCCCCTC GAATCACTTT TCCCGGCCTG 600 (2) INFORMATION FOR SEQ ID NO:4:
(i) S~u~ CHARACTERISTICS:
(A) LENGTH: 378 amino acids (B) TYPE: amino acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~Qu~ DESCRIPTION: SEQ ID NO:4:
J~ f~-J~ ~P
CA 0223138~ 1998-03-06 p ~ L ~ 6 94g4 ~ S F ~ 7 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg Gly Ser Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala Hi8 Glu Arg Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn Ser Ser Ala Ser Asn Ala Ser ~ CA 0223l38~ l998-03-06 PC~ 9~/l4~6~
~19494 I~S 16 SEP l9g7 (2) 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 Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln 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 Ly~
Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg Gly Ser Met Ala Ser Ser Gly Met Ala Asp Ser Ala Asn His Leu Pro Phe Phe Phe Gly Asn Ile Thr Arg Glu Glu Ala Glu Asp Tyr Leu Val Gln Gly Gly Met Ser Asp Gly Leu Tyr Leu Leu Arg Gln Ser Arg Asn Tyr Leu Gly Gly Phe Ala Leu Ser Val Ala His Gly Arg Lys Ala His His Tyr Thr Ile Glu Arg Glu Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Arg Thr His Ala Ser Pro Ala Asp Leu Cys His Tyr His Ser Gln Glu Ser Asp Gly Leu Val Cys Leu Leu Lys Lys Pro Phe Asn Arg Pro Gln Gly Val Gln Pro Lys Thr Gly Pro Phe Glu Asp Leu Lys Glu Asn Leu Ile Arg Glu Tyr Val Lys Gln Thr Trp Asn Leu Gln Gly Gln Ala Leu Glu Gln Ala Ile Ile Ser Gln Lys Pro Gln Leu Glu Lys Leu Ile Ala Thr Thr Ala His Glu Lys Met Pro Trp Phe His Gly Lys Ile Ser Arg Glu Glu Ser Glu Gln Ile Val Leu Ile Gly Ser Lys Thr Asn . CA 0223l38~ l998-03-06 PC~ 4 ~ ./ 1 4 ~ ~ 3 19494 1 ~ 1 6 SEP lg97 Gly Lys Phe Leu Ile Arg Ala Arg Asp Asn Asn Gly Ser Tyr Ala Leu Cys Leu Leu His Glu Gly Lys Val Leu His Tyr Arg Ile Asp Lys Asp Lys Thr Gly Lys Leu Ser Ile Pro Glu Gly Lys Lys Phe Asp Thr Leu Trp Gln Leu Val Glu His Tyr Ser Tyr Lys Ala Asp Gly Leu Leu Arg Val Leu Thr Val Pro Cys Gln Lys Ile Gly Thr Gln Gly Asn Val Asn (2) INFORMATION FOR SEQ ID NO:6:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 224 amino acids . Y (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~UU~N~ DESCRIPTION: SEQ ID NO:6:
Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Al~ Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg Gly Ser Met Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe Phe Lys Asn Leu Ser Arg Ly~ Asp Ala Glu Arg Gln Leu Leu Ala Pro Gly Asn Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gln Asn Gln Gly Glu Val Val Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr Ile Ser Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys Gln Thr , CA 0223138S 1998-03-06 P~Tlll~ 9 ~ ~14~6~
.1~494 lP ~ S ~ ~ ~EP 1997 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gly Leu Val Pro Arg Gly Ser l 5 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANn~.nNR.~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~U~N~'~ DESCRIPTION: SEQ ID NO:8:
(2) INFORMATION FOR SEQ ID NO:9:
, ~ ~
~Q~N~ CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDRr)NRc:s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:9:
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 0223l385 l998-03-06 p ~ ~s.
'19494 1 ~ S 1 6 SEP 1997 (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) sTR~NnEnNF~s: single (D) TOPOLOGY: linear f '~ ~
.. (ii) MOhECUhE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) sTRs~Nn~nNF~s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~QukN~ DESCRIPTION: SEQ ID NO:12:
(2) INFORMATION FOR SEQ ID NO:13:
U~N~ CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:13:
~D~0 .
~ CA 0223l385 l998-03-06 --'I9~9~ -22- ~P ~ S 1 6 SEP 1997 (2) INFORMATION FOR SEQ ID NO:14:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEUU~N~ DESCRIPTION: SEQ ID NO:15:
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
~ (A) NAME/KEY: Other (B) LOCATION: 6...6 (D) OTHER INFORMATION: Xaa = Phosphorylated Tyrosine (A) NAME/KEY: Other -~B) LOCATION: 17...17 (D) OTHER INFORMATION: Xaa = Phosphorylated Tyrosine (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:16:
M~NDEDSHEr , -~ CA 0223l385 l998-03-06 P~ ;96/1~6~
19494 }~ ~ S l fi S~P 1g~7 Gly Ser Asn Gln Leu Xaa Asn Glu Leu Asn Leu Gly Ary Arg Glu Glu Xaa Asp Val Leu Asp Lys (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANnFnNF~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ,~ (ix) FEATURE:
(A) NAME/KEY: Other (B) LOCATION: 4...4 (D) OTHER INFORMATION: Xaa = Phosphorylated Tyrosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Glu Pro Gln Xaa Glu Glu Ile Pro Ile Tyr Leu FN~=SllEr
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1137 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
? GAAGAAGGGG TTGCCCAGAT GA~l~l~G~l CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240 TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC ~ C~T~llc 300 GCTCACCTGC C~ll~ll'~lA CGGCAGCATC TCGCGTGCCG AGGCCGAGGA GCACCTGAAG 420 AA~llC~l~C TGAGGCCGCG GAAGGAGCAG GGCACATACG CC~l~lCCCT CATCTATGGG 960 '- (2) INFORMATION FOR SEQ ID No:2:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 1155 base pairs (B) TYPE: nucleic acid (C) STRANI~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~u~ DESCRIPTION: SEQ ID NO:2:
ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCAC~llCCC CAAGCGCGGC 60 TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC ~ C~l~llC 300 ~ CA 0223l38~ l998-03-06 PC~ q~l456~
IP~WS16SEP1997 TCAAAGACAA ATGGAAAGTT CCTGATCCGA GCCA~A~-A~A ACAACGGCTC CTACGCCCTG 960 TATAAAGCAG A~ AAGAGTTCTT ACTGTCCCAT GTCAAAAAAT CGGCACACAG 1140 (2) INFORMATION FOR SEQ ID NO:3:
(i) ~QU~Nt_~ CHARACTERISTICS:
(A) LENGTH: 675 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAA~AAA~T TGATTCCTCC 120 TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC ~l~t_l~C~lt_llC 300 GAGCCCGAAC C~ CAAGAACCTG AGCCGCAAGG ACGCGGAGCG GCAGCTCCTG 420 GCGC'CCG~A ACACTCACGG ~lC~llCCTC ATCCGGGAGA GC~A~A~CAC CGCGGGATCG 480 ATCCGTAATC TG~A~AACGG TGG~ll~lAC ATCTCCCCTC GAATCACTTT TCCCGGCCTG 600 (2) INFORMATION FOR SEQ ID NO:4:
(i) S~u~ CHARACTERISTICS:
(A) LENGTH: 378 amino acids (B) TYPE: amino acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~Qu~ DESCRIPTION: SEQ ID NO:4:
J~ f~-J~ ~P
CA 0223138~ 1998-03-06 p ~ L ~ 6 94g4 ~ S F ~ 7 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg Gly Ser Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala Hi8 Glu Arg Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn Ser Ser Ala Ser Asn Ala Ser ~ CA 0223l38~ l998-03-06 PC~ 9~/l4~6~
~19494 I~S 16 SEP l9g7 (2) 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 Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln 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 Ly~
Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg Gly Ser Met Ala Ser Ser Gly Met Ala Asp Ser Ala Asn His Leu Pro Phe Phe Phe Gly Asn Ile Thr Arg Glu Glu Ala Glu Asp Tyr Leu Val Gln Gly Gly Met Ser Asp Gly Leu Tyr Leu Leu Arg Gln Ser Arg Asn Tyr Leu Gly Gly Phe Ala Leu Ser Val Ala His Gly Arg Lys Ala His His Tyr Thr Ile Glu Arg Glu Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Arg Thr His Ala Ser Pro Ala Asp Leu Cys His Tyr His Ser Gln Glu Ser Asp Gly Leu Val Cys Leu Leu Lys Lys Pro Phe Asn Arg Pro Gln Gly Val Gln Pro Lys Thr Gly Pro Phe Glu Asp Leu Lys Glu Asn Leu Ile Arg Glu Tyr Val Lys Gln Thr Trp Asn Leu Gln Gly Gln Ala Leu Glu Gln Ala Ile Ile Ser Gln Lys Pro Gln Leu Glu Lys Leu Ile Ala Thr Thr Ala His Glu Lys Met Pro Trp Phe His Gly Lys Ile Ser Arg Glu Glu Ser Glu Gln Ile Val Leu Ile Gly Ser Lys Thr Asn . CA 0223l38~ l998-03-06 PC~ 4 ~ ./ 1 4 ~ ~ 3 19494 1 ~ 1 6 SEP lg97 Gly Lys Phe Leu Ile Arg Ala Arg Asp Asn Asn Gly Ser Tyr Ala Leu Cys Leu Leu His Glu Gly Lys Val Leu His Tyr Arg Ile Asp Lys Asp Lys Thr Gly Lys Leu Ser Ile Pro Glu Gly Lys Lys Phe Asp Thr Leu Trp Gln Leu Val Glu His Tyr Ser Tyr Lys Ala Asp Gly Leu Leu Arg Val Leu Thr Val Pro Cys Gln Lys Ile Gly Thr Gln Gly Asn Val Asn (2) INFORMATION FOR SEQ ID NO:6:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 224 amino acids . Y (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) ~UU~N~ DESCRIPTION: SEQ ID NO:6:
Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Al~ Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg Gly Ser Met Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe Phe Lys Asn Leu Ser Arg Ly~ Asp Ala Glu Arg Gln Leu Leu Ala Pro Gly Asn Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala Gly Ser Phe Ser Leu Ser Val Arg Asp Phe Asp Gln Asn Gln Gly Glu Val Val Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr Ile Ser Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His Tyr Thr Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys Gln Thr , CA 0223138S 1998-03-06 P~Tlll~ 9 ~ ~14~6~
.1~494 lP ~ S ~ ~ ~EP 1997 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gly Leu Val Pro Arg Gly Ser l 5 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANn~.nNR.~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~U~N~'~ DESCRIPTION: SEQ ID NO:8:
(2) INFORMATION FOR SEQ ID NO:9:
, ~ ~
~Q~N~ CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDRr)NRc:s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:9:
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 0223l385 l998-03-06 p ~ ~s.
'19494 1 ~ S 1 6 SEP 1997 (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) sTR~NnEnNF~s: single (D) TOPOLOGY: linear f '~ ~
.. (ii) MOhECUhE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) sTRs~Nn~nNF~s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~QukN~ DESCRIPTION: SEQ ID NO:12:
(2) INFORMATION FOR SEQ ID NO:13:
U~N~ CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:13:
~D~0 .
~ CA 0223l385 l998-03-06 --'I9~9~ -22- ~P ~ S 1 6 SEP 1997 (2) INFORMATION FOR SEQ ID NO:14:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEUU~N~ DESCRIPTION: SEQ ID NO:15:
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
~ (A) NAME/KEY: Other (B) LOCATION: 6...6 (D) OTHER INFORMATION: Xaa = Phosphorylated Tyrosine (A) NAME/KEY: Other -~B) LOCATION: 17...17 (D) OTHER INFORMATION: Xaa = Phosphorylated Tyrosine (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:16:
M~NDEDSHEr , -~ CA 0223l385 l998-03-06 P~ ;96/1~6~
19494 }~ ~ S l fi S~P 1g~7 Gly Ser Asn Gln Leu Xaa Asn Glu Leu Asn Leu Gly Ary Arg Glu Glu Xaa Asp Val Leu Asp Lys (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANnFnNF~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ,~ (ix) FEATURE:
(A) NAME/KEY: Other (B) LOCATION: 4...4 (D) OTHER INFORMATION: Xaa = Phosphorylated Tyrosine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Glu Pro Gln Xaa Glu Glu Ile Pro Ile Tyr Leu FN~=SllEr
Claims (14)
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.
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 [3H]- or [125I]-labeled 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: SH1, 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.
11. 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 11, 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.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US382495P | 1995-09-15 | 1995-09-15 | |
| US60/003,824 | 1995-09-15 | ||
| GBGB9603486.3A GB9603486D0 (en) | 1996-02-20 | 1996-02-20 | A high throughputassay using fusion proteins |
| GB9603486.3 | 1996-02-20 | ||
| PCT/US1996/014563 WO1997010502A1 (en) | 1995-09-15 | 1996-09-11 | A high throughput assay using fusion proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2231385A1 true CA2231385A1 (en) | 1997-03-20 |
Family
ID=26308762
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002231385A Abandoned CA2231385A1 (en) | 1995-09-15 | 1996-09-11 | A high throughput assay using fusion proteins |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0855029A4 (en) |
| JP (1) | JP2000501171A (en) |
| CA (1) | CA2231385A1 (en) |
| WO (1) | WO1997010502A1 (en) |
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|---|---|---|---|---|
| US6187757B1 (en) | 1995-06-07 | 2001-02-13 | Ariad Pharmaceuticals, Inc. | Regulation of biological events using novel compounds |
| WO1998041866A1 (en) * | 1997-03-14 | 1998-09-24 | Merck & Co., Inc. | A high throughput assay using fusion proteins |
| AU1121000A (en) * | 1998-10-19 | 2000-05-08 | Ariad Gene Therapeutics, Inc. | Materials and methods involving conditional retention domains |
| US7067526B1 (en) | 1999-08-24 | 2006-06-27 | Ariad Gene Therapeutics, Inc. | 28-epirapalogs |
| BRPI0210598B8 (en) | 2001-06-22 | 2021-05-25 | Hoffmann La Roche | processes for producing a soluble complex comprising a target amyloidogenic protein and a peptidyl prolyl isomerase chaperone, for producing a soluble chaperone surface retroviral glycoprotein complex, and for detecting at least one antibody to a retroviral glycoprotein of surface in a sample, as well as soluble complex, and composition |
| ATE457716T1 (en) | 2002-12-30 | 2010-03-15 | Angiotech Int Ag | RELEASE OF ACTIVE INGREDIENTS FROM QUICK-GELLING POLYMER COMPOSITION |
| EP1892530A1 (en) * | 2006-08-25 | 2008-02-27 | Boehringer Ingelheim Pharma GmbH & Co. KG | Method for determining transport activity of a transport protein |
| WO2009089549A1 (en) | 2008-01-11 | 2009-07-16 | Massachusetts Eye & Ear Infirmary | Conditional-stop dimerizable caspase transgenic animals |
| EP3663405A1 (en) | 2013-06-11 | 2020-06-10 | Takara Bio USA, Inc. | Protein enriched microvesicles and methods of making and using the same |
Family Cites Families (1)
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|---|---|---|---|---|
| DE3483099D1 (en) * | 1984-03-15 | 1990-10-04 | Immunex Corp | TEST FOR IMMEDIATELY DETECTING LIGANDS, TEST SET AND ITS PRODUCTION. |
-
1996
- 1996-09-11 JP JP9512067A patent/JP2000501171A/en active Pending
- 1996-09-11 WO PCT/US1996/014563 patent/WO1997010502A1/en not_active Application Discontinuation
- 1996-09-11 EP EP96931529A patent/EP0855029A4/en not_active Withdrawn
- 1996-09-11 CA CA002231385A patent/CA2231385A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0855029A4 (en) | 2000-03-08 |
| JP2000501171A (en) | 2000-02-02 |
| WO1997010502A1 (en) | 1997-03-20 |
| EP0855029A1 (en) | 1998-07-29 |
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