CA2231330A1 - A high throughput assay using fusion proteins - Google Patents
A high throughput assay using fusion proteins Download PDFInfo
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- CA2231330A1 CA2231330A1 CA 2231330 CA2231330A CA2231330A1 CA 2231330 A1 CA2231330 A1 CA 2231330A1 CA 2231330 CA2231330 CA 2231330 CA 2231330 A CA2231330 A CA 2231330A CA 2231330 A1 CA2231330 A1 CA 2231330A1
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- fusion protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
Abstract
This application describes a high throughput assay for screening for compounds capable of binding to a fusion protein which consists of a target protein and an FK506-binding protein. The method for preparing the DNA encoding for the fusion protein and for expressing that DNA is also described in the application. The invention also discloses the recombinant DNA and protein sequences for several fusion proteins.
Description
9~ PC~T/US96/14567 U~S 19 SEP 19~7 TITLE OF THE INVENTION
A HIGH THROUGHPUT ASSAY USING FUSION PROTEINS
BACK;GROUND OF THE INVENTION
Src homology 2 (SH2) domains are a family of homologous protein domains that share the common property of recogni~in,~r 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 [125I]Bolton-Hunter reagent. In both cases, the -- isotop~es 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 separiation of bound complex from free phosphopeptide by washing of the glutathione-Sepharose beads This is a nonequilibrium procedure that riisks 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 AIUEI'~DED ~~ET
s ~ nr~1l)S 9 6 ~1 4 5 6 7 ~US 19 SEP 1997 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 5 al., Proc. Natl. Acad. Sci. U.S.A., 92, 3199-3203 (1995); Lemmon et al., Biochemist~y, 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.
--' 10 SUMMARY OF THE INVENTION
The instant invention covers a method of screening for compounds capable of binding to a fusion protein which comprises combiining a test compound, a tagged ligand, a fusion protein (target 15 protein, peptide linker and FK506-binding protein), and a radiolabeled ligand~ in a coated microscintillation plate, and then measuring the scinti]lation counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a contro assay in the absence of the test compound, so as to determine the effect 20 the test compound has on the binding of the tagged ligand. Also within -- the scope of this invention are the processes for preparing and expressing the recombinant DNA encoding a fusion protein. This invention further relates to the recombinant DNA expression vector capable of expressing the fusion protein. This invention further relates 25 to a process for purifying the recombinant fusion protein. This invention provides an immediate means of making use of micrc,scintillation plate 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 30 invention does not require specialized radiochemical synthesis and is readi]y adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors.
~MENDED StlEET
CA 02231330 1998-03-06 ~ r ~ , 19.j~ IP~S 1 9 SEP 1997 BRIEl~ DESCRIPTION OF THE FIGURES
Figure 1.
A.) Binding of the streptavidin microscintillation plate, 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 DESCRIPTION OF THE INVENTION
-~ 10 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, and a radiolabeled ligand;
b) ~lflin~ the mixture to a coated microscintillation plate;
c) incubating the mixture for between about 1 hour and about 24 hours;
d) measuring the plate-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 e) 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 second embodiment of this invention is a process for preparing a recombinant DNA expression vector encoding for a fusion protein comprising the steps of:
a) removing the stop codon on DNA encoding for an FK506-binding protein;
b) synthesizing a modified DNA fragment on the DNA encoding for the FK506-binding protein which encodes for a peptide linker;
,r!~,3 CL~LI-~
~S 19 SÉP 1~97 c) digesting an expression vector at cloning sites;
d) cloning the modified DNA fragment encoding for the FK506-binding protein with a peptide linker into the digested expression vector to generate a recombinant DNA
expression vector encoding for FK506-binding protein with a peptide linker; and e) cloning DNA encoding for a target protein into a recombinant DNA expression vector encoding for FK506-binding protein with a peptide linker to produce the recombinant --- 10 DNA expression vector encoding for the fusion protein.
A third embodiment of this invention is a process for expressing recombinant DNA encoding for a fusion protein in an expression vector comprising the steps of:
a) transforming a host cell with the fusion protein expression vector;
b) inducing expression of the fusion protein in the host cell;
c) recovering the fusion protein from the host cell; and d) purifying the fusion protein.
A fourth embodiment of this invention is a process for purifying an isolated FBP-SH2 fusion protein, comprising the steps of:
a) preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support;
b) preparing a freeze/thaw extract from cells expressing the fusion protein;
c) loading the extract onto the affinity matrix and washing off unbound protein; and d) eluting the desired fusion protein with phenyl phosphate.
The terrn "fusion protein" refers to a "target protein" fused to an "FK506-binding protein" (FKBP), the two proteins being separated by a "peptide linker".
9~ P C T/U S 9 6 / 1 4 5 6 7 ~P~JS 19 SEP 19g7 A "peptide linker" may consist of a sequence cont~ininp from about 1 to about 20 arnino 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 5 sequence GLVPRGS (SEQ.ID.NO. 7).
The terrn "target protein" refers to any protein that has a defineld ligand. Included within this definition of target protein are single and multiple signal transduction domains, such as, but not lirnited to, Src homology 1 (SH1), Src homology 2 (SH2), Src homology 3 - 10 (SH3). and pleckstrin homology (PH) domains [Hanks & Hunter, FASEirs J., 9, 576-596 (1995); Bolen, Curr. Opin. lmmunol., 7, 306-311- (1995); Kuriyan & Cowburn, Curr. Opin. Struct. Biol., 3, 828-837 (1993)l; Cohen et al., Cell, 80, 237-248 (1995)]. The term "SH1 domain" refers to a farnily of homologous protein domains that bind 15 ATP and catalyze tyrosine phosphorylation of peptide and protein substrates. The term "SH2 domain" refers to a farnily of homologous protein domains that share the cornrnon property of recognizing phosp]horylated tyrosine residues in specific peptide contexts. The term "SH3 domain" refers to a family of homologous protein domains that 20 share the cornrnon 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.
Exam~ples of SH2 domains which may be utilized in the method of the invention include, but are not limited to, the single and tandem SH2 25 domains present in the tyrosine kinases ZAP, SYK and LCK. The DNA
seque]nces were obtained from GenBank, National Center for Biotechnology Inforrnation, 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 30 LCK (X13529). The sequences for ZAP, SYK and LCK are disclosed in the sequence listing as follows: the isolated DNA encoding for a fusion protein containing ZAP is (SEQ.ID.NO.1); the isolated DNA
encod~ing for a fusion protein containing SYK is (SEQ.ID.NO.2); the isolated DNA encoding for a fusion protein cont~ining LCK is ~ ~ .r (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 FKBP-LCK:SH2 fusion protein is (SEQ.ID.NO.6).
The terrn "tagged ligand" refers to a biotinylated or epitope tagged ligand for the target protein.
The term "radiolabeled ligand" refers to a [3H]-, [125I]-, [14C]-, [35S]-, [32p], or [33P]-labeled ligand which binds to the FKBP.
An example of a radiolabeled ligand useful in the instant invention is - 10 [3H]-dihydroFK506.
The terrn "coated microscintillation plates" refers to strept;avidin-coated microscintillation plates when the tagged ligand is biotinylated, and to anti-epitope antibody bound to anti-antibody-coated or protein A-coated microscintillation plates when the tagged ligand is epitope-tagged. Examples of coated microscintillation plates useful in the instant invention are streptavidin-coated, sheep anti-rabbit-coated, and goat anti-mouse-coated FlashPlate Plus (DuPont-NEN). Additional coatings, including but not limited to protein A, may be applied to uncoated FlashPlates by methods known to those skilled in the art.
The term "control assay" refers to the assay when performed in the presence of the tagged ligand, the fusion protein, the radio]Labeled ligand and the coated microscintill~tion plates, but in the absence of the test compound.
The term FK506-binding proteins may include, but are not 25 limited to, the below listed FKBPs and FKBP homologues, which include a citation to the references which disclose them. This list is not intenlded to limit the scope of the invention.
l~n-m~li?n FKB~P-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).
AMEND~D SHE~
S' 1 ~ S~~ 199~/
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 B~-lghm~n et al., Mol. Cell. Biol., 8, 4395-4402(1995) .
FKBP-52 Galat et al., supra.
Bacte ria 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 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.
Fun~al 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 limited to, bacteria, yeast, bluegreen algae, plant cells, insect cells and ~nim~l cells.
r ~ t~
~95'~ ' ~'J f~ f 7 7 r ~ 7 IPEA/US 1.~ ~FP 1997 E7cpression 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 ~nimAl cells.
Specifically designed vectors allow the shuttling of DNA
between hosts such as bacteria-yeast or bacteria-~nim~l cells. An appropriately constructed expression vector may contain: an origin of ' 10 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 F~BP fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pl7 (USB), pET (Novagen), pIBI (IBI), pProEX-1 (Gibco/BRL), pBluescript iI (Stratagene), pTZl~R 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 cont~inin~ 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 95'~ fj 7 J
resulting supernatant contains >80% pure FKBP fusion, which if desired can be purified further by conventional methods. Alterna~ively, the assay is not dependent on pure protein and the initial periplasmic preparation may be used directly. A thrombin site located between S FKBF' 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 10 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.
After a suitable incubation period to allow complex formation to occur, the mixture is transferred to a coated microscintillation plate to capture 20 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 in the microscintillation plate 25 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 in Figure 1.
The present invention can be understood further by the following examples, which do not constitute a limitation of the 30 invention.
195~ ~ J
~ 9 9 7 Process for Preparing the FKBP fusion clonin~ vector General techniques for modifying and expressing genes in 5 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 -~- 10 sequence encoding a thrombin site (Leu-Val-Pro-Arg) and BamHI
restricltion site (GAAl-rC) were amplified using the polymerase chain reaetion (PCR). The PCR reaction contained the following primers:5'-GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' (SEQ..[D.NO.8); and 5'-TACGAATTCTGGCGTGGATCCACGC
15 GGAACCAGACCTTCCAGTTTTAG-3' (SEQ.ID.NO.9); 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 Esche~richia coli cells. Clones cont~inin~ an insert were identified using 20 PCR with fl~nking vector primers. Dideoxy DNA sequencing confirlmed 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 transformed with the 25 ligation mixture, and colonies containing the insert were identified using PCR with primers encoding for fl~nking vector sequences. The FKBP
fusion cloning vector is called pET9dFKBPt.
Process for Preparin~ 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 l:hat the reading frame was conserved with that of FKBP in the 35 fusion vector. At the 3'-end, the fragment also incorporated a stop A~ENDE~ SHEEt 9 6 l 1 4 5 6 7 SI~ P 1997 codon followed by a BamHI site. The PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
S '-ATTAGGATCCATGCCAGATCCTGCAGCTCACCTGCCCT-3' (SEQ.ID.NO.10) and 5'-ATATGGATCCTTACCAGAGGCGTTGCT-3' (SEQ.ID.NO.11). The fragment was cloned into a suitable vector, sequenced, digested with BamHI, and the insert cont~ininp the SH2 domains ligated to BamHI treated pET9dFKBPt, and transformed into E. coli. Clones containing inserts in the correct orientation were identi~led by PCR or restriction enzyme analysis. Plasmid DNA was ~~~10 prepared and used to transform BL21(DE3) cells.
..
Process for Preparin~ the FK-SYK fusion expression vector The expression vector for the tandem SH2 domains of SYK
fused to FKBP was prepared as in Exarnple 2 except that the PCR
reacti~n contained Raji cell cDNA (Clontech) and the following primers: 5'-CAATAGGATCCATGGCCAGCAGCGGCATGGCTGA-3' (SEQ,ID.NO.12) and 5'-GACCTAGGATCCCTAATTAACATTTCC
CTGTGTGCCGAT-3' (SEQ.ID.NO.13).
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 Example 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
5'-ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3' (SEQ.ID.NO.14) and 5'-ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTG
TGCA-3' (SEQ.ID.NO.15).
PCT/US 9h/14567 lPEAlUS 19 SEP 1997 FK-ZAP
S Step A: Process for Expression of FK-ZAP
E. coli BL21(DE3) cells cont~ining the pET9dFKBPt/ ZAP
SH2 plasmid were grown in Luria-Bertani (LB) media containing 50 microgram/ml k~n~mycin at about 37 degrees C until the optical density measured at 600 nm was about 0.5-1Ø Expression of the FK-ZAP
-- 10 fusion protein was induced with 0.1 mM isopropyl beta-- thiogalactopyranoside and the cells were grown for another 3-5 hr at about 30 degrees C. They were pelleted at 4400 x g for about 10 min at about 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, 15 pepstatin, leupeptin, and bestatin. The resuspended pellet was frozen at about -20 degrees C until further purification.
Step B: Process for Purification of FK-ZAP
The affinity matrix for purification of FK-ZAP was 20 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.16) and washing out unbound peptide. Frozen cells cont~ining FK-ZAP were thawed in warm water, refrozen on dry ice 25 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
30 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.
195~ 7 ~ _~
- ' ~ ?~
FK-SYK
E. coli BL21(DE3) cells containing the pET9dFKBPt/
5 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.
~'. 10 - 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 puri~lcation of FK-LCK was prepared by combining agarose-immobilized avidin with excess biotinyl- EPQpYEEIPIYL (SEQ.ID.NO.17) and washing out unbound peptidLe. The rem~ining methodology for puri~lcation was the same as Example 5.
Assay of phosphopeptide binding to FK-ZAP
Assays were conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM Dl-r, 0.01% TWEEN-20, pH 7Ø
300 Ill of a mixture of buffer and varying amounts of biotinyl-phosphopeptide were combined with 25 ,ul of FK-ZAP protein and 50 ,ul of [3H]-dihydroFK506 (DuPont NEN) in microfuge tubes. A 150 111 portion of each assay was then transferred to the well of a streptavidin-coated FlashPlate Plus (DuPont-NEN) and an additional 50 111 of buffer was added. Final concentrations of the assay components were:
0-50 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK
(SEQ.ID.NO. 16) 100 nM FK-ZAP fusion protein 25 nM [3H]-dihydroFK506 . . ~,r - ' ' ~ ,7 195'~
~ t ~ 9q7~
The plate was sealed and incubated 20 hours. Plate-bound radioactivity was measured at various timepoints in a Packard Topcount microplate scintillation counter.
Method of Screening for Antagonists of FK-ZAP
Assays are conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01% TWEEN-20, pH 7Ø
-~- 10 10 ~11 of a DMSO solution of test compound(s) and 120 111 of biotinyl-phosphopeptide stock solution are dispensed into the wells of a standard 96-well plate. Next, 20 ~11 of a mixture of FK-ZAP protein and [3H]-dihydroFK506 (DuPont NEN) are added to each test well. The assays are then transferred to the wells of a streptavidin-coated FlashPlate (DuPont NEN). Final concentrations of the assay components are:
25 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK
(SEQ.ID.NO. 16) 25 nM FK-ZAP fusion protein 10 nM [3H]-dihydroFK506 5% DMSO
The plate is sealed and incubated between 1 and 8 hours. Bead-bound radioactivity is then measured in a Packard Topcount microplate scintill~ion counter.
Method of Screenin~ for Anta~onists of FK-SYK
The assays are conducted as set forth in Example 9, except that FK-SYK replaces FK-ZAP.
~ O S~
19'i~ 9 ~ / 1 4 5 6 7 1 9 SirP 1997 Method of Screening for Antagonists of FK-LCK
The assays are conducted as set forth in Example 9, except S that FK-LCK replaces FK-ZAP and the tagged ligand is 25 nM
biotinyl-EPQpYEEIPIYL (SEQ.ID.NO.17).
CA 0223l330 l998-03-06 l~524 PCT/US 9 6 / 14 5 6 7 -16- IPE~/US 19 SEP 1997 SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: MERCK & CO., INC.
(ii) TITLE OF THE INVENTION: A HIGH THROUGHPUT ASSAY USING
FUSION PROTEINS
(iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE 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) ~IP: 07065-0900 (v) COMPUTER READABLE 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: PCT/US96/14567 (B) FILING DATE: 11-SEP-1996 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) US APPLICATION NUMBER: 60/003.819 FILING DATE: 15 SEPTEMBER 1995 (B) GB APPLICATION NUMBER: 9605210.5 FILING DATE: 12 MARCH 1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Camara, Valerie J
(B) REGISTRATION NUMBER: 35,090 (C) REFERENCE/DOCKET NUMBER: 19524 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-3902 (B) TELEFAX: 908-594-4720 (C) TELEX:
;, .,,, ~,, CA 0223l330 l998-03-06 ~T~VS 9 k / I 4 5 6 7 19.~'4 ~ v v, (2) INFORMATION FOR SEQ ID NO:1:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CAGACC'TGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 120 CGGGAC'AGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 180 GAAGA~GGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240 GATGTC,GAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGCC AGATCCTGCA 360 AACGG('ACCT ACGCCATTGC CGGCGGCAAA GCGCACTGTG GACCGGCAGA GCTCTGCGAG 600 TTCTA('TCGC GCGACCCCGA CGGGCTGCCC TGCAACCTGC GCAAGCCGTG CAACCGGCCG 660 TCGGG('CTCG AGCCGCAGCC GGGGGTCTTC GACTGCCTGC GAGACGCCAT GGTGCGTGAC 720 TACGT(,CGCC AGACGTGGAA GCTGGAGGGC GAGGCCCTGG AGCAGGCCAT CATCAGCCAG 780 GCCCC(,CAGG TGGAGAAGCT CATTGCTACG ACGGCCCACG AGCGGATGCC CTGGTACCAC 840 (2) INFORMATION FOR SEQ ID NO:2:
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CA 0223l330 l998-03-06 -18- ~ EP I~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATGG(,AGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60 ATGC('TTGGT TCCATGGAAA AATCTCTCGG GAAGAATCTG AGCAAATTGT CCTGATAGGA 900 CTCT('CATCC CCGAGGGAAA GAAGTTCGAC ACGCTCTGGC AGCTAGTCGA GCATTATTCT 1080 (2) INFORMATION FOR SEQ ID NO:3:
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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 C;ln 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 I.eu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg C,ly 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 His 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 ~ Li CA 02231330 1998-03-06 ~ , , '' t ~~
19~24 -20- IPEA~lJS 1 9 SEP 1997 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 (2) INFORMATION FOR SEQ ID NO:5:
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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 C;ln 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 l?he 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 ~ , , ~ ; . . ~ ;
CA 02231330 l998-03-06 l9.524 , ~r~ h 7 Ser Arg Glu Glu Ser Glu Gln Ile Val Leu Ile Gly Ser Lys Thr Asn 2~0 295 300 Gly L~s 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:
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Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro L,ys 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 C:ln 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 (,ly Ser Met Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro Gly Asn :L30 135 140 Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala Gly Ser Phe ',er 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 19.~24 ~ 5 S 7 -22- ~ t5~ i (2) INFORMATION FOR SEQ ID NO:7:
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Gly Leu Val Pro Arg Gly Ser (2) INFORMATION FOR SEQ ID NO:8:
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(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
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(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
AMEN-DED SltEET
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(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
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(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
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(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 0223l330 1998-03-06 PCT/US ~ ~ / '4 567 - 24 ~ A/Us 1 9 SEP 199~
(ii) MOLECULE TYPE: Genomic DNA
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(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) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Gly Ser Asn Gln Leu Xaa Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Xaa Asp Val Leu Asp Lys (2) INFORMATION FOR SEQ ID NO:17:
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(ii) MOLECULE ~-YPE: 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 l 5 l0 AMENDED SHEET
A HIGH THROUGHPUT ASSAY USING FUSION PROTEINS
BACK;GROUND OF THE INVENTION
Src homology 2 (SH2) domains are a family of homologous protein domains that share the common property of recogni~in,~r 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 [125I]Bolton-Hunter reagent. In both cases, the -- isotop~es 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 separiation of bound complex from free phosphopeptide by washing of the glutathione-Sepharose beads This is a nonequilibrium procedure that riisks 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 AIUEI'~DED ~~ET
s ~ nr~1l)S 9 6 ~1 4 5 6 7 ~US 19 SEP 1997 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 5 al., Proc. Natl. Acad. Sci. U.S.A., 92, 3199-3203 (1995); Lemmon et al., Biochemist~y, 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.
--' 10 SUMMARY OF THE INVENTION
The instant invention covers a method of screening for compounds capable of binding to a fusion protein which comprises combiining a test compound, a tagged ligand, a fusion protein (target 15 protein, peptide linker and FK506-binding protein), and a radiolabeled ligand~ in a coated microscintillation plate, and then measuring the scinti]lation counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a contro assay in the absence of the test compound, so as to determine the effect 20 the test compound has on the binding of the tagged ligand. Also within -- the scope of this invention are the processes for preparing and expressing the recombinant DNA encoding a fusion protein. This invention further relates to the recombinant DNA expression vector capable of expressing the fusion protein. This invention further relates 25 to a process for purifying the recombinant fusion protein. This invention provides an immediate means of making use of micrc,scintillation plate 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 30 invention does not require specialized radiochemical synthesis and is readi]y adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors.
~MENDED StlEET
CA 02231330 1998-03-06 ~ r ~ , 19.j~ IP~S 1 9 SEP 1997 BRIEl~ DESCRIPTION OF THE FIGURES
Figure 1.
A.) Binding of the streptavidin microscintillation plate, 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 DESCRIPTION OF THE INVENTION
-~ 10 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, and a radiolabeled ligand;
b) ~lflin~ the mixture to a coated microscintillation plate;
c) incubating the mixture for between about 1 hour and about 24 hours;
d) measuring the plate-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 e) 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 second embodiment of this invention is a process for preparing a recombinant DNA expression vector encoding for a fusion protein comprising the steps of:
a) removing the stop codon on DNA encoding for an FK506-binding protein;
b) synthesizing a modified DNA fragment on the DNA encoding for the FK506-binding protein which encodes for a peptide linker;
,r!~,3 CL~LI-~
~S 19 SÉP 1~97 c) digesting an expression vector at cloning sites;
d) cloning the modified DNA fragment encoding for the FK506-binding protein with a peptide linker into the digested expression vector to generate a recombinant DNA
expression vector encoding for FK506-binding protein with a peptide linker; and e) cloning DNA encoding for a target protein into a recombinant DNA expression vector encoding for FK506-binding protein with a peptide linker to produce the recombinant --- 10 DNA expression vector encoding for the fusion protein.
A third embodiment of this invention is a process for expressing recombinant DNA encoding for a fusion protein in an expression vector comprising the steps of:
a) transforming a host cell with the fusion protein expression vector;
b) inducing expression of the fusion protein in the host cell;
c) recovering the fusion protein from the host cell; and d) purifying the fusion protein.
A fourth embodiment of this invention is a process for purifying an isolated FBP-SH2 fusion protein, comprising the steps of:
a) preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support;
b) preparing a freeze/thaw extract from cells expressing the fusion protein;
c) loading the extract onto the affinity matrix and washing off unbound protein; and d) eluting the desired fusion protein with phenyl phosphate.
The terrn "fusion protein" refers to a "target protein" fused to an "FK506-binding protein" (FKBP), the two proteins being separated by a "peptide linker".
9~ P C T/U S 9 6 / 1 4 5 6 7 ~P~JS 19 SEP 19g7 A "peptide linker" may consist of a sequence cont~ininp from about 1 to about 20 arnino 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 5 sequence GLVPRGS (SEQ.ID.NO. 7).
The terrn "target protein" refers to any protein that has a defineld ligand. Included within this definition of target protein are single and multiple signal transduction domains, such as, but not lirnited to, Src homology 1 (SH1), Src homology 2 (SH2), Src homology 3 - 10 (SH3). and pleckstrin homology (PH) domains [Hanks & Hunter, FASEirs J., 9, 576-596 (1995); Bolen, Curr. Opin. lmmunol., 7, 306-311- (1995); Kuriyan & Cowburn, Curr. Opin. Struct. Biol., 3, 828-837 (1993)l; Cohen et al., Cell, 80, 237-248 (1995)]. The term "SH1 domain" refers to a farnily of homologous protein domains that bind 15 ATP and catalyze tyrosine phosphorylation of peptide and protein substrates. The term "SH2 domain" refers to a farnily of homologous protein domains that share the cornrnon property of recognizing phosp]horylated tyrosine residues in specific peptide contexts. The term "SH3 domain" refers to a family of homologous protein domains that 20 share the cornrnon 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.
Exam~ples of SH2 domains which may be utilized in the method of the invention include, but are not limited to, the single and tandem SH2 25 domains present in the tyrosine kinases ZAP, SYK and LCK. The DNA
seque]nces were obtained from GenBank, National Center for Biotechnology Inforrnation, 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 30 LCK (X13529). The sequences for ZAP, SYK and LCK are disclosed in the sequence listing as follows: the isolated DNA encoding for a fusion protein containing ZAP is (SEQ.ID.NO.1); the isolated DNA
encod~ing for a fusion protein containing SYK is (SEQ.ID.NO.2); the isolated DNA encoding for a fusion protein cont~ining LCK is ~ ~ .r (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 FKBP-LCK:SH2 fusion protein is (SEQ.ID.NO.6).
The terrn "tagged ligand" refers to a biotinylated or epitope tagged ligand for the target protein.
The term "radiolabeled ligand" refers to a [3H]-, [125I]-, [14C]-, [35S]-, [32p], or [33P]-labeled ligand which binds to the FKBP.
An example of a radiolabeled ligand useful in the instant invention is - 10 [3H]-dihydroFK506.
The terrn "coated microscintillation plates" refers to strept;avidin-coated microscintillation plates when the tagged ligand is biotinylated, and to anti-epitope antibody bound to anti-antibody-coated or protein A-coated microscintillation plates when the tagged ligand is epitope-tagged. Examples of coated microscintillation plates useful in the instant invention are streptavidin-coated, sheep anti-rabbit-coated, and goat anti-mouse-coated FlashPlate Plus (DuPont-NEN). Additional coatings, including but not limited to protein A, may be applied to uncoated FlashPlates by methods known to those skilled in the art.
The term "control assay" refers to the assay when performed in the presence of the tagged ligand, the fusion protein, the radio]Labeled ligand and the coated microscintill~tion plates, but in the absence of the test compound.
The term FK506-binding proteins may include, but are not 25 limited to, the below listed FKBPs and FKBP homologues, which include a citation to the references which disclose them. This list is not intenlded to limit the scope of the invention.
l~n-m~li?n FKB~P-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).
AMEND~D SHE~
S' 1 ~ S~~ 199~/
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 B~-lghm~n et al., Mol. Cell. Biol., 8, 4395-4402(1995) .
FKBP-52 Galat et al., supra.
Bacte ria 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 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.
Fun~al 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 limited to, bacteria, yeast, bluegreen algae, plant cells, insect cells and ~nim~l cells.
r ~ t~
~95'~ ' ~'J f~ f 7 7 r ~ 7 IPEA/US 1.~ ~FP 1997 E7cpression 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 ~nimAl cells.
Specifically designed vectors allow the shuttling of DNA
between hosts such as bacteria-yeast or bacteria-~nim~l cells. An appropriately constructed expression vector may contain: an origin of ' 10 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 F~BP fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pl7 (USB), pET (Novagen), pIBI (IBI), pProEX-1 (Gibco/BRL), pBluescript iI (Stratagene), pTZl~R 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 cont~inin~ 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 95'~ fj 7 J
resulting supernatant contains >80% pure FKBP fusion, which if desired can be purified further by conventional methods. Alterna~ively, the assay is not dependent on pure protein and the initial periplasmic preparation may be used directly. A thrombin site located between S FKBF' 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 10 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.
After a suitable incubation period to allow complex formation to occur, the mixture is transferred to a coated microscintillation plate to capture 20 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 in the microscintillation plate 25 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 in Figure 1.
The present invention can be understood further by the following examples, which do not constitute a limitation of the 30 invention.
195~ ~ J
~ 9 9 7 Process for Preparing the FKBP fusion clonin~ vector General techniques for modifying and expressing genes in 5 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 -~- 10 sequence encoding a thrombin site (Leu-Val-Pro-Arg) and BamHI
restricltion site (GAAl-rC) were amplified using the polymerase chain reaetion (PCR). The PCR reaction contained the following primers:5'-GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' (SEQ..[D.NO.8); and 5'-TACGAATTCTGGCGTGGATCCACGC
15 GGAACCAGACCTTCCAGTTTTAG-3' (SEQ.ID.NO.9); 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 Esche~richia coli cells. Clones cont~inin~ an insert were identified using 20 PCR with fl~nking vector primers. Dideoxy DNA sequencing confirlmed 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 transformed with the 25 ligation mixture, and colonies containing the insert were identified using PCR with primers encoding for fl~nking vector sequences. The FKBP
fusion cloning vector is called pET9dFKBPt.
Process for Preparin~ 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 l:hat the reading frame was conserved with that of FKBP in the 35 fusion vector. At the 3'-end, the fragment also incorporated a stop A~ENDE~ SHEEt 9 6 l 1 4 5 6 7 SI~ P 1997 codon followed by a BamHI site. The PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
S '-ATTAGGATCCATGCCAGATCCTGCAGCTCACCTGCCCT-3' (SEQ.ID.NO.10) and 5'-ATATGGATCCTTACCAGAGGCGTTGCT-3' (SEQ.ID.NO.11). The fragment was cloned into a suitable vector, sequenced, digested with BamHI, and the insert cont~ininp the SH2 domains ligated to BamHI treated pET9dFKBPt, and transformed into E. coli. Clones containing inserts in the correct orientation were identi~led by PCR or restriction enzyme analysis. Plasmid DNA was ~~~10 prepared and used to transform BL21(DE3) cells.
..
Process for Preparin~ the FK-SYK fusion expression vector The expression vector for the tandem SH2 domains of SYK
fused to FKBP was prepared as in Exarnple 2 except that the PCR
reacti~n contained Raji cell cDNA (Clontech) and the following primers: 5'-CAATAGGATCCATGGCCAGCAGCGGCATGGCTGA-3' (SEQ,ID.NO.12) and 5'-GACCTAGGATCCCTAATTAACATTTCC
CTGTGTGCCGAT-3' (SEQ.ID.NO.13).
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 Example 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
5'-ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3' (SEQ.ID.NO.14) and 5'-ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTG
TGCA-3' (SEQ.ID.NO.15).
PCT/US 9h/14567 lPEAlUS 19 SEP 1997 FK-ZAP
S Step A: Process for Expression of FK-ZAP
E. coli BL21(DE3) cells cont~ining the pET9dFKBPt/ ZAP
SH2 plasmid were grown in Luria-Bertani (LB) media containing 50 microgram/ml k~n~mycin at about 37 degrees C until the optical density measured at 600 nm was about 0.5-1Ø Expression of the FK-ZAP
-- 10 fusion protein was induced with 0.1 mM isopropyl beta-- thiogalactopyranoside and the cells were grown for another 3-5 hr at about 30 degrees C. They were pelleted at 4400 x g for about 10 min at about 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, 15 pepstatin, leupeptin, and bestatin. The resuspended pellet was frozen at about -20 degrees C until further purification.
Step B: Process for Purification of FK-ZAP
The affinity matrix for purification of FK-ZAP was 20 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.16) and washing out unbound peptide. Frozen cells cont~ining FK-ZAP were thawed in warm water, refrozen on dry ice 25 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
30 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.
195~ 7 ~ _~
- ' ~ ?~
FK-SYK
E. coli BL21(DE3) cells containing the pET9dFKBPt/
5 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.
~'. 10 - 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 puri~lcation of FK-LCK was prepared by combining agarose-immobilized avidin with excess biotinyl- EPQpYEEIPIYL (SEQ.ID.NO.17) and washing out unbound peptidLe. The rem~ining methodology for puri~lcation was the same as Example 5.
Assay of phosphopeptide binding to FK-ZAP
Assays were conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM Dl-r, 0.01% TWEEN-20, pH 7Ø
300 Ill of a mixture of buffer and varying amounts of biotinyl-phosphopeptide were combined with 25 ,ul of FK-ZAP protein and 50 ,ul of [3H]-dihydroFK506 (DuPont NEN) in microfuge tubes. A 150 111 portion of each assay was then transferred to the well of a streptavidin-coated FlashPlate Plus (DuPont-NEN) and an additional 50 111 of buffer was added. Final concentrations of the assay components were:
0-50 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK
(SEQ.ID.NO. 16) 100 nM FK-ZAP fusion protein 25 nM [3H]-dihydroFK506 . . ~,r - ' ' ~ ,7 195'~
~ t ~ 9q7~
The plate was sealed and incubated 20 hours. Plate-bound radioactivity was measured at various timepoints in a Packard Topcount microplate scintillation counter.
Method of Screening for Antagonists of FK-ZAP
Assays are conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01% TWEEN-20, pH 7Ø
-~- 10 10 ~11 of a DMSO solution of test compound(s) and 120 111 of biotinyl-phosphopeptide stock solution are dispensed into the wells of a standard 96-well plate. Next, 20 ~11 of a mixture of FK-ZAP protein and [3H]-dihydroFK506 (DuPont NEN) are added to each test well. The assays are then transferred to the wells of a streptavidin-coated FlashPlate (DuPont NEN). Final concentrations of the assay components are:
25 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK
(SEQ.ID.NO. 16) 25 nM FK-ZAP fusion protein 10 nM [3H]-dihydroFK506 5% DMSO
The plate is sealed and incubated between 1 and 8 hours. Bead-bound radioactivity is then measured in a Packard Topcount microplate scintill~ion counter.
Method of Screenin~ for Anta~onists of FK-SYK
The assays are conducted as set forth in Example 9, except that FK-SYK replaces FK-ZAP.
~ O S~
19'i~ 9 ~ / 1 4 5 6 7 1 9 SirP 1997 Method of Screening for Antagonists of FK-LCK
The assays are conducted as set forth in Example 9, except S that FK-LCK replaces FK-ZAP and the tagged ligand is 25 nM
biotinyl-EPQpYEEIPIYL (SEQ.ID.NO.17).
CA 0223l330 l998-03-06 l~524 PCT/US 9 6 / 14 5 6 7 -16- IPE~/US 19 SEP 1997 SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: MERCK & CO., INC.
(ii) TITLE OF THE INVENTION: A HIGH THROUGHPUT ASSAY USING
FUSION PROTEINS
(iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE 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) ~IP: 07065-0900 (v) COMPUTER READABLE 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: PCT/US96/14567 (B) FILING DATE: 11-SEP-1996 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) US APPLICATION NUMBER: 60/003.819 FILING DATE: 15 SEPTEMBER 1995 (B) GB APPLICATION NUMBER: 9605210.5 FILING DATE: 12 MARCH 1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Camara, Valerie J
(B) REGISTRATION NUMBER: 35,090 (C) REFERENCE/DOCKET NUMBER: 19524 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-3902 (B) TELEFAX: 908-594-4720 (C) TELEX:
;, .,,, ~,, CA 0223l330 l998-03-06 ~T~VS 9 k / I 4 5 6 7 19.~'4 ~ v v, (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CAGACC'TGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 120 CGGGAC'AGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 180 GAAGA~GGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240 GATGTC,GAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGCC AGATCCTGCA 360 AACGG('ACCT ACGCCATTGC CGGCGGCAAA GCGCACTGTG GACCGGCAGA GCTCTGCGAG 600 TTCTA('TCGC GCGACCCCGA CGGGCTGCCC TGCAACCTGC GCAAGCCGTG CAACCGGCCG 660 TCGGG('CTCG AGCCGCAGCC GGGGGTCTTC GACTGCCTGC GAGACGCCAT GGTGCGTGAC 720 TACGT(,CGCC AGACGTGGAA GCTGGAGGGC GAGGCCCTGG AGCAGGCCAT CATCAGCCAG 780 GCCCC(,CAGG TGGAGAAGCT CATTGCTACG ACGGCCCACG AGCGGATGCC CTGGTACCAC 840 (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: Genomic DNA
CA 0223l330 l998-03-06 -18- ~ EP I~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATGG(,AGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60 ATGC('TTGGT TCCATGGAAA AATCTCTCGG GAAGAATCTG AGCAAATTGT CCTGATAGGA 900 CTCT('CATCC CCGAGGGAAA GAAGTTCGAC ACGCTCTGGC AGCTAGTCGA GCATTATTCT 1080 (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: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AMENDED SilEET
CA 02231330 l998-03-06 19.524 ' ~ S~ 7 -19- ~PE~JUS 1 9 SEP 1~97 (2) INFOR~ATION FOR SEQ ID NO: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 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 C;ln 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 I.eu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro Arg C,ly 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 His 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 ~ Li CA 02231330 1998-03-06 ~ , , '' t ~~
19~24 -20- IPEA~lJS 1 9 SEP 1997 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 (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 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 C;ln 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 l?he 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 ~ , , ~ ; . . ~ ;
CA 02231330 l998-03-06 l9.524 , ~r~ h 7 Ser Arg Glu Glu Ser Glu Gln Ile Val Leu Ile Gly Ser Lys Thr Asn 2~0 295 300 Gly L~s 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) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro L,ys 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 C:ln 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 (,ly Ser Met Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe Phe Lys Asn Leu Ser Arg Lys Asp Ala Glu Arg Gln Leu Leu Ala Pro Gly Asn :L30 135 140 Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala Gly Ser Phe ',er 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 19.~24 ~ 5 S 7 -22- ~ t5~ i (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 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
AMEN-DED SltEET
CA 0223l330 l998-03-06 l9524 PCT/US 9 h / ' 4 5 6 7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
. ,. ~
- (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 0223l330 1998-03-06 PCT/US ~ ~ / '4 567 - 24 ~ A/Us 1 9 SEP 199~
(ii) MOLECULE TYPE: Genomic DNA
(xi3 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 ~D3 TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE 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) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Gly Ser Asn Gln Leu Xaa Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Xaa Asp Val Leu Asp Lys (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear l9524 -- 25 ~ v ~
(ii) MOLECULE ~-YPE: 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 l 5 l0 AMENDED SHEET
Claims (47)
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, and a radiolabeled ligand;
b) adding the mixture to a coated microscintillation plate;
c) incubating the mixture for between about 1 hour and about 24 hours;
d) measuring the plate-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 e) 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, and a radiolabeled ligand;
b) adding the mixture to a coated microscintillation plate;
c) incubating the mixture for between about 1 hour and about 24 hours;
d) measuring the plate-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 e) 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 the coated microscintillation plates 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 a [3H]-, [125I]-, [14C], [35S]-, [32p], or [33P]-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 for 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.
15. A process for preparing a recombinant DNA
expression vector encoding for a fusion protein comprising the steps of:
a) removing the stop codon on DNA encoding for an FK506-binding protein;
b) synthesizing a modified DNA fragment on the DNA encoding for the FK506-binding protein which encodes for a peptide linker;
c) digesting an expression vector at cloning sites;
d) cloning the modified DNA fragment encoding for the FK506-binding protein with a peptide linker into the digested expression vector to generate a recombinant DNA
expression vector encoding for FK506-binding protein with a peptide linker; and e) cloning DNA encoding for a target protein into a recombinant DNA expression vector encoding for FK506-binding protein with a peptide linker to produce the recombinant DNA expression vector encoding for the fusion protein.
expression vector encoding for a fusion protein comprising the steps of:
a) removing the stop codon on DNA encoding for an FK506-binding protein;
b) synthesizing a modified DNA fragment on the DNA encoding for the FK506-binding protein which encodes for a peptide linker;
c) digesting an expression vector at cloning sites;
d) cloning the modified DNA fragment encoding for the FK506-binding protein with a peptide linker into the digested expression vector to generate a recombinant DNA
expression vector encoding for FK506-binding protein with a peptide linker; and e) cloning DNA encoding for a target protein into a recombinant DNA expression vector encoding for FK506-binding protein with a peptide linker to produce the recombinant DNA expression vector encoding for the fusion protein.
16. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 15, wherein the target protein is a single or multiple signal transduction domain.
expression vector encoding the fusion protein, as recited in Claim 15, wherein the target protein is a single or multiple signal transduction domain.
17. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 16, wherein the single or multiple signal transduction domain is selected from the group consisting of: SH1, SH2, SH3 and PH domains.
expression vector encoding the fusion protein, as recited in Claim 16, wherein the single or multiple signal transduction domain is selected from the group consisting of: SH1, SH2, SH3 and PH domains.
18. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 17, wherein the single or multiple signal transduction domain is an SH2 domain.
expression vector encoding the fusion protein, as recited in Claim 17, wherein the single or multiple signal transduction domain is an SH2 domain.
19. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 18, wherein the single or multiple signal transduction domain is an SH2 domain selected from the group consisting of ZAP:SH2, SYK:SH2 and LCK:SH2.
expression vector encoding the fusion protein, as recited in Claim 18, wherein the single or multiple signal transduction domain is an SH2 domain selected from the group consisting of ZAP:SH2, SYK:SH2 and LCK:SH2.
20. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 19, wherein the FK506-binding protein is a 12 kDa FK506 binding protein.
expression vector encoding the fusion protein, as recited in Claim 19, wherein the FK506-binding protein is a 12 kDa FK506 binding protein.
21. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 20, wherein the peptide linker has the amino acid sequence GLVPRGS
(SEQ.ID.NO.7).
expression vector encoding the fusion protein, as recited in Claim 20, wherein the peptide linker has the amino acid sequence GLVPRGS
(SEQ.ID.NO.7).
22. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 21, wherein the target protein is ZAP:SH2.
expression vector encoding the fusion protein, as recited in Claim 21, wherein the target protein is ZAP:SH2.
23. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 21, wherein the target protein is SYK:SH2.
expression vector encoding the fusion protein, as recited in Claim 21, wherein the target protein is SYK:SH2.
24. The process for preparing a recombinant DNA
expression vector encoding the fusion protein, as recited in Claim 21, wherein the target protein is LCK:SH2.
expression vector encoding the fusion protein, as recited in Claim 21, wherein the target protein is LCK:SH2.
25. Isolated DNA encoding for a fusion protein comprising the sequence:
(SEQ.ID.NO.1).
(SEQ.ID.NO.1).
26. Isolated DNA encoding for a fusion protein comprising the sequence:
(SEQ.ID.NO. 2).
(SEQ.ID.NO. 2).
27. Isolated DNA encoding for a fusion protein comprising the sequence:
(SEQ.ID.NO. 3).
(SEQ.ID.NO. 3).
28. A FKBP-ZAP:SH2 fusion protein comprising the sequence:
(SEQ. ID. NO.4).
(SEQ. ID. NO.4).
29. A FKBP-SYK:SH2 fusion protein comprising the sequence:
(SEQ. ID. NO. 5).
(SEQ. ID. NO. 5).
30. A FKBP-LCK:SH2 fusion protein comprising the sequence:
(SEQ. ID. NO. 6).
(SEQ. ID. NO. 6).
31. A process for expressing recombinant DNA encoding for a fusion protein in an expression vector comprising the steps of:
a) transforming a host cell with the fusion protein expression vector;
b) inducing expression of the fusion protein in the host cell;
c) recovering the fusion protein from the host cell; and d) purifying the fusion protein.
a) transforming a host cell with the fusion protein expression vector;
b) inducing expression of the fusion protein in the host cell;
c) recovering the fusion protein from the host cell; and d) purifying the fusion protein.
32. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 31, wherein the target protein is a single or multiple signal transduction domain.
encoding a fusion protein, as recited in Claim 31, wherein the target protein is a single or multiple signal transduction domain.
33. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 32, wherein the single or multiple signal transduction domain is selected from the group consisting of: SH1, SH2, SH3 and PH domains.
encoding a fusion protein, as recited in Claim 32, wherein the single or multiple signal transduction domain is selected from the group consisting of: SH1, SH2, SH3 and PH domains.
34. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 33, wherein the single or multiple signal transduction domain is a single or multiple SH2 domain.
encoding a fusion protein, as recited in Claim 33, wherein the single or multiple signal transduction domain is a single or multiple SH2 domain.
35. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 34, wherein the single or multiple SH2 domain is selected from a group consisting of ZAP:SH2, SYK:SH2 and LCK:SH2.
encoding a fusion protein, as recited in Claim 34, wherein the single or multiple SH2 domain is selected from a group consisting of ZAP:SH2, SYK:SH2 and LCK:SH2.
36. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 35, wherein the FK506-binding protein is human 12kDa FK506-binding protein.
encoding a fusion protein, as recited in Claim 35, wherein the FK506-binding protein is human 12kDa FK506-binding protein.
37. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 36, wherein the host cell is from bacteria, yeast, blue green algae, plant cells, insect cells, or animal cells.
encoding a fusion protein, as recited in Claim 36, wherein the host cell is from bacteria, yeast, blue green algae, plant cells, insect cells, or animal cells.
38. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 37, wherein the host cell is an E.coli strain selected from a group consisting of BL21 (DE3), Nova Blue (DE3), and JM109 (DE3).
encoding a fusion protein, as recited in Claim 37, wherein the host cell is an E.coli strain selected from a group consisting of BL21 (DE3), Nova Blue (DE3), and JM109 (DE3).
39. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 38, wherein the single or multiple SH2 domain is ZAP:SH2.
encoding a fusion protein, as recited in Claim 38, wherein the single or multiple SH2 domain is ZAP:SH2.
40. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 38, wherein the single or multiple SH2 domain is SYK:SH2.
encoding a fusion protein, as recited in Claim 38, wherein the single or multiple SH2 domain is SYK:SH2.
41. The process for expressing recombinant DNA
encoding a fusion protein, as recited in Claim 38, wherein the single or multiple SH2 domain is LCK:SH2.
encoding a fusion protein, as recited in Claim 38, wherein the single or multiple SH2 domain is LCK:SH2.
42. The process for purifying an isolated FKBP-SH2 fusion protein comprising the steps of:
a) preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support;
b) preparing a freeze/thaw extract from cells expressing the fusion protein;
c) loading the extract onto the affinity matrix and washing off unbound protein; and d) eluting the desired fusion protein with phenyl phosphate.
a) preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support;
b) preparing a freeze/thaw extract from cells expressing the fusion protein;
c) loading the extract onto the affinity matrix and washing off unbound protein; and d) eluting the desired fusion protein with phenyl phosphate.
43. A recombinant FKBP-SH2 domain T7 RNA
polymerase-based expression vector, wherein the DNA encodes for the FKBP-ZAP:SH2 fusion protein and has the DNA sequence (SEQ. ID. NO. 1).
polymerase-based expression vector, wherein the DNA encodes for the FKBP-ZAP:SH2 fusion protein and has the DNA sequence (SEQ. ID. NO. 1).
44. A recombinant FKBP-SH2 domain T7 RNA
polymerase-based expression vector, wherein the DNA encodes for the FKBP-SYK:SH2 fusion protein and has the DNA sequence (SEQ. ID. NO. 2).
polymerase-based expression vector, wherein the DNA encodes for the FKBP-SYK:SH2 fusion protein and has the DNA sequence (SEQ. ID. NO. 2).
45. A recombinant FKBP-SH2 domain T7 RNA
polymerase-based expression vector, wherein the DNA encodes for the FKBP-LCK:SH2 fusion protein and has the DNA sequence (SEQ. ID. NO. 3).
polymerase-based expression vector, wherein the DNA encodes for the FKBP-LCK:SH2 fusion protein and has the DNA sequence (SEQ. ID. NO. 3).
46. A recombinant host cell containing the recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector wherein the recombinant host cell isselected from the group consisting of: E. coli BL21 (DE3), E. coli Nova Blue (DE3), and E. coli JM109 (DE3).
47. The recombinant host cell containing the recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector as recited in claim 46, wherein the recombinant host cell is E.
coli BL21 (DE3).
coli BL21 (DE3).
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US381995P | 1995-09-15 | 1995-09-15 | |
| US60/003,819 | 1995-09-15 | ||
| GB9605210.5 | 1996-03-12 | ||
| GBGB9605210.5A GB9605210D0 (en) | 1996-03-12 | 1996-03-12 | A high throughput assay using fusion proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2231330A1 true CA2231330A1 (en) | 1997-03-20 |
Family
ID=26308918
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2231330 Abandoned CA2231330A1 (en) | 1995-09-15 | 1996-09-11 | A high throughput assay using fusion proteins |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0871648A1 (en) |
| JP (1) | JPH11513246A (en) |
| CA (1) | CA2231330A1 (en) |
| WO (1) | WO1997010253A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997039326A2 (en) * | 1996-04-18 | 1997-10-23 | Ariad Pharmaceuticals, Inc. | In vitro fluorescence polarization assay |
| US6962982B2 (en) | 2001-06-22 | 2005-11-08 | Roche Diagnostics Corporation | Soluble complexes of target proteins and peptidyl prolyl isomerase chaperones and methods of making and using them |
| US7094757B2 (en) | 2001-06-22 | 2006-08-22 | Roche Diagnostics Corporation | Complexes comprising a prion protein and a peptidyl prolyl isomerase chaperone, and method for producing and using them |
| JP4168028B2 (en) * | 2002-06-25 | 2008-10-22 | 積水化学工業株式会社 | Expression vector, host, fusion protein, method for producing fusion protein, and method for producing protein |
| ATE527345T1 (en) | 2006-01-03 | 2011-10-15 | Hoffmann La Roche | CHIMERIC FUSION PROTEIN WITH SUPERIOR CHAPERONE AND FOLDING ACTIVITIES |
| CN114651061A (en) * | 2019-10-23 | 2022-06-21 | 施特丁·奈德兰卡克研究所-安东尼·范·列文虎克医院 | Chimeric polypeptides for modulating immune cells |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69230433D1 (en) * | 1991-01-18 | 2000-01-20 | Univ New York | CDNS CLONING METHOD FOR RECIPE TYROSINKINASE TARGET PROTEIN AND HGRB PROTEINS |
| CA2054602C (en) * | 1991-10-31 | 2003-04-22 | Anthony Pawson | Method for assaying for a substance that affects an sh2-phosphorylated ligand regulatory system |
| US5498597A (en) * | 1992-01-17 | 1996-03-12 | Dana-Farber Cancer Institute, Inc. | FKBP-13, an FK506-binding immunophilin |
| GB9210176D0 (en) * | 1992-05-12 | 1992-06-24 | Cemu Bioteknik Ab | Chemical method |
| US5464745A (en) * | 1993-03-31 | 1995-11-07 | Novagen, Inc. | Protein ligand binding region mapping system |
| US5580979A (en) * | 1994-03-15 | 1996-12-03 | Trustees Of Tufts University | Phosphotyrosine peptidomimetics for inhibiting SH2 domain interactions |
-
1996
- 1996-09-11 EP EP96935808A patent/EP0871648A1/en not_active Withdrawn
- 1996-09-11 JP JP9512071A patent/JPH11513246A/en active Pending
- 1996-09-11 WO PCT/US1996/014567 patent/WO1997010253A1/en not_active Application Discontinuation
- 1996-09-11 CA CA 2231330 patent/CA2231330A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0871648A1 (en) | 1998-10-21 |
| JPH11513246A (en) | 1999-11-16 |
| WO1997010253A1 (en) | 1997-03-20 |
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