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WO1997010253A1 - A high throughput assay using fusion proteins - Google Patents

A high throughput assay using fusion proteins Download PDF

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Publication number
WO1997010253A1
WO1997010253A1 PCT/US1996/014567 US9614567W WO9710253A1 WO 1997010253 A1 WO1997010253 A1 WO 1997010253A1 US 9614567 W US9614567 W US 9614567W WO 9710253 A1 WO9710253 A1 WO 9710253A1
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WIPO (PCT)
Prior art keywords
fusion protein
recited
protein
expression vector
binding
Prior art date
Application number
PCT/US1996/014567
Other languages
French (fr)
Inventor
Alice Marcy
Scott P. Salowe
Douglas Wisniewski
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from GBGB9605210.5A external-priority patent/GB9605210D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to JP9512071A priority Critical patent/JPH11513246A/en
Priority to EP96935808A priority patent/EP0871648A1/en
Publication of WO1997010253A1 publication Critical patent/WO1997010253A1/en

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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.)
    • 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
    • G01N33/68—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

Definitions

  • Src homology 2 (SH2) domains are a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts. They have routinely been expressed in E. coli as fusion proteins with glutathione-S-transferase (GST). This usually provides high level expression and straightforward affinity purification on glutathione- Sepharose. Ligand binding is then assayed by incubating the GST/SH2 with a radiolabeled phosphopeptide, precipitating the complex with glutathione-Sepharose, washing the beads, and then counting the beads to determine bound radioactivity [Isakov et al., J. Exp.
  • 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.
  • 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), and a radiolabeled ligand in a coated microscintillation plate, and then measuring the scintillation counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a control assay in the absence of the test compound, so as to determine the effect the test compound has on the binding of the tagged ligand.
  • Also within die 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 to a process for purifying the recombinant fusion protein.
  • This invention provides an immediate means of making use of microscintillation 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 invention does not require specialized radiochemical synthesis and is readily adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors. BRIEF DESCRIPTION OF THE FIGURES Figure 1.
  • 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) 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 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; 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.
  • 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 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.
  • fusion protein refers to a "target protein” fused to an "FK506-binding protein” (FKBP), the two proteins being separated by a "peptide linker".
  • a “peptide linker” may consist of a sequence containing from about 1 to about 20 amino acids, which may or may not include the sequence for a protease cleavage site.
  • An example of a peptide linker which is a protease cleavage site is represented by the amino acid sequence GLPRGS.
  • target protein refers to any protein that has a defined ligand. Included within this definition of target protein are single and multiple signal transduction domains, such as, but not limited to, Src homology 1 (SHI), Src homology 2 (SH2), Src homology 3 (SH3), and pleckstrin homology (PH) domains [Hanks & Hunter,
  • SHI domain refers to a family of homologous protein domains that bind ATP and catalyze tyrosine phosphorylation of peptide and protein substrates.
  • SH2 domain refers to a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts.
  • SH3 domain refers to a family of homologous protein domains that share the common property of recognizing polyproline type II helices.
  • PH domain refers to a family of homologous protein domains that mediate both protein-protein and protein-lipid interactions.
  • SH2 domains which may be utilized in the method of the invention include, but are not limited to, the single and tandem SH2 domains present in the tyrosine kinases ZAP, SYK and LCK.
  • the DNA sequences were obtained from GenBank, National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894. The Accession Numbers for the sequences are: human ZAP (L05148); human SYK (L28824) and human LCK (X I 3529).
  • tagged ligand refers to a biotinylated or epitope tagged ligand for the target protein.
  • radiolabeled ligand refers to a H]-, [125j]_ [ 14 C ]., [35s]-, [32p]_, or [33 P ]_ labe led ligand which binds to the FKBP.
  • An example of a radiolabeled ligand useful in the instant invention is [3H]-dihydroFK506.
  • coated microscintillation plates refers to streptavidin-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.
  • 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.
  • control assay refers to the assay when performed in the presence of the tagged ligand, the fusion protein, the radiolabeled ligand and the coated microscintillation plates, but in the absence of the test compound.
  • FK506-binding proteins may include, but are not limited to, the below listed FKBPs and FKBP homologues, which include a citation to the references which disclose them. This list is not intended to limit the scope of the invention.
  • host cells include, but are not limited to, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express genes in a variety of host cells, such as, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
  • An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • vectors suitable for FKBP fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pT7 (USB), pET (Novagen), pIBI (IB I), pProEX-1 (Gibco/BRL), pBluescript II (Stratagene), pTZ18R and pTZ19R (USB), pSE420 (Invitrogen), pVL1392 (Invitrogen), pBlueBac (Invitrogen), pBAcPAK (Clontech), pHIL (Invitrogen), pYES2 (Invitrogen), pCDNA (Invitrogen), pREP (Invitrogen) or the like.
  • the expression vector may be introduced into host cells via any one of a number of techinques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation.
  • E. coli containing an expression plasmid with the target gene fused to FKBP are grown and appropriately induced. The cells are then pelleted and resuspended in a suitable buffer.
  • 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 supernatant contains >80% pure FKBP fusion, which if desired can be purified further by conventional methods. Altematively, the assay is not dependent on pure protein and the initial periplasmic preparation may be used directly.
  • a t-hrombin site located between FKBP and the target protein can be used as a means to cleave FKBP from the fusion; such cleaved material may be a suitable negative control for subsequent assays.
  • a fusion protein which contains a single or multiple SH2 domain(s) may be purified by preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support. A freeze/thaw extract is prepared from the cells which express the fusion protein and is loaded onto the affinity matrix. The desired fusion protein is then specifically eluted with phenyl phosphate.
  • 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 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 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 PCR reaction contained the following primers :5'- GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' and 5 - TACGAATTCTGGCGTGGATCCACGCGGAACCAGACCTTCCAGT TTTAG-3' and a plasmid containing human FKBP-12 as the template.
  • the resulting 367 base pair amplification product was ligated into the vector pCRII (Invitrogen) and the ligation mixture transformed into competent Escherichia coli cells. Clones containing an insert were identified using PCR with flanking vector primers. Dideoxy DNA sequencing confirmed the nucleotide sequence of one positive isolate.
  • the altered 338 base pair FKBP fragment was excised from the pCRII plasmid using Ncol and BamHI and ligated into Ncol and/f ⁇ mHI digested pET9d (Novagen) plasmid. Competent E. coli were transformed with the ligation mixture, and colonies containing the insert were identified using PCR with primers encoding for flanking vector sequences.
  • the FKBP fusion cloning vector is called pET9dFKBPt.
  • 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 inco ⁇ orated a stop codon followed by a BamHI site.
  • the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
  • the expression vector for the tandem SH2 domains of Syk fused to FKBP was prepared as in Example 2 except that the PCR reaction contained Raji cell cDNA (Clontech) and the following primers: 5 -CAATAGGATCCATGGCCAGCAGCGGCATGGCTGA-3' and 5 -GACCTAGGATCCCTAATTAACATTTCCCTGTGTGCCGAT- 3 * .
  • 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:
  • Step A Process for Expression of FK-ZAP
  • E. coli BL21 (DE3) cells containing the pET9dFKBPt/ ZapSH2 plasmid were grown in Luria-Bertani (LB) media containing 50 microgram/ml kanamycin at about 37 degrees C until the optical density measured at 600 nm was about 0.5-1.0.
  • Expression of the FK-ZAP fusion protein was induced with 0.1 mM isopropyl beta- thiogalactopyranoside and the cells were grown for another 3-5 hr at about 30 degrees C.
  • Step B Process for Purification of FK-ZAP
  • the affinity matrix for purification of FK-ZAP was prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the ⁇ l ITAM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK, and washing out unbound peptide. Frozen cells containing FK-ZAP were thawed in warm water, refrozen on dry ice for about 25 min., then thawed again.
  • 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.
  • E. coli BL21 (DE3) cells containing the pET9dFKBPt/ LckSH2 plasmid were grown, induced, and harvested as described in Example 5.
  • the affinity matrix for purification of FK-LCK was prepared by combining agarose-immobilized avidin with excess biotinyl- EPQpYEEIPIYL, and washing out unbound peptide. The remaining methodology for purification was the same as Example 5.
  • Assays were conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01 % TWEEN-20, pH 7.0. 300 ⁇ l of a mixture of buffer and varying amounts of biotinyl- phosphopeptide were combined with 25 ⁇ l of FK-ZAP protein and 50 ⁇ l of [3H]-dihydroFK506 (DuPont NEN) in microfuge tubes. A 150 ⁇ l portion of each assay was then transferred to the well of a streptavidin- coated FlashPlate Plus (DuPont-NEN) and an additional 50 ⁇ l of buffer was added. Final concentrations of the assay components were:
  • Assays are conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01 % TWEEN-20, pH 7.0. 10 ⁇ l of a DMSO solution of test compound(s) and 120 ⁇ l of biotinyl- phosphopeptide stock solution are dispensed into the wells of a standard 96-well plate. Next, 20 ⁇ l of a mixture of FK-ZAP protein and [ 3 H]-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
  • the assays are conducted as set forth in Example 9, except that FK-SYK replaces FK-ZAP.
  • the assays are conducted as set forth in Example 9, except that FK-LCK replaces FK-ZAP and the tagged ligand is 25 nM biotinyl-EPQpYEEIPIYL.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GAGCCCGAAC CCTGGTTCTT CAAGAACCTG AGCCGCAAGG ACGCGGAGCG GCAGCTCCTG 420
  • ATCCGTAATC TGGACAACGG TGGCTTCTAC ATCTCCCCTC GAATCACTTT TCCCGGCCTG 600

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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

TITLE QF THE INVENTION
A HIGH THROUGHPUT ASSAY USING FUSION PROTEINS
BACKGROUND OF THE INVENTION Src homology 2 (SH2) domains are a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts. They have routinely been expressed in E. coli as fusion proteins with glutathione-S-transferase (GST). This usually provides high level expression and straightforward affinity purification on glutathione- Sepharose. Ligand binding is then assayed by incubating the GST/SH2 with a radiolabeled phosphopeptide, precipitating the complex with glutathione-Sepharose, washing the beads, and then counting the beads to determine bound radioactivity [Isakov et al., J. Exp. Med., 181 , 375- 380 (1995); Piccione et al., Biochemistry, 32, 3197-3202 (1993); Huyer et nl, 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 [125r]B0it0n-Hunter reagent. In both cases, the isotopes are short-lived and thus require frequent preparation of material. In the case of enzymatic phosphorylation, the appropriate kinase must also be available in sufficient quantity to generate enough material for screening purposes. Second, the protocol requires separation of bound complex from free phosphopeptide by washing of the glutathione-Sepharose beads. This is a nonequilibrium procedure that risks dissociation of the bound ligand, particularly when off-rates are fast. Thus, there is the possibility of misleading results. Finally, due to the number of manipulations and centrifugations involved, the protocol is very tedious to conduct manually and is not readily adaptable to robotic automation to increase throughput.
Two additional methods for measuring the interaction of proteins and ligands that have been applied to SH2 domains are biospecific interaction analysis using surface plasmon resonance and isothermal titration calorimetry (Felder et al., Mol. Cell. Biol., 13, 1449-1455 (1993); Panayotou et al., Mol. Cell. Biol., 13, 3567-3576 ( 1993); Payne et al., Proc. Natl. Acad. Sci. U.S. A., 90, 4902-4906 (1993); Morelock et al., J. Med. Chem. 38, 1309-18 (1995); Ladbury et al., Proc. Natl. Acad. Sci. U.S.A., 92, 3199-3203 (1995); 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.
SUMMARY OF THE INVENΗON
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), and a radiolabeled ligand in a coated microscintillation plate, and then measuring the scintillation counts attributable to the binding of the tagged ligand to the fusion protein in the presence of the test compound relative to a control assay in the absence of the test compound, so as to determine the effect the test compound has on the binding of the tagged ligand. Also within die 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 to a process for purifying the recombinant fusion protein. This invention provides an immediate means of making use of microscintillation 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 invention does not require specialized radiochemical synthesis and is readily adaptable to robotic automation for high capacity screening for agonists, antagonists, and/or inhibitors. BRIEF 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 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) 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 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; 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.
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 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.
The term "fusion protein" refers to a "target protein" fused to an "FK506-binding protein" (FKBP), the two proteins being separated by a "peptide linker". A "peptide linker" may consist of a sequence containing from about 1 to about 20 amino acids, which may or may not include the sequence for a protease cleavage site. An example of a peptide linker which is a protease cleavage site is represented by the amino acid sequence GLPRGS.
The term "target protein" refers to any protein that has a defined ligand. Included within this definition of target protein are single and multiple signal transduction domains, such as, but not limited to, Src homology 1 (SHI), Src homology 2 (SH2), Src homology 3 (SH3), and pleckstrin homology (PH) domains [Hanks & Hunter,
FASEB J., 9, 576-596 (1995); Bolen, Curr. Opin. Immunol., 1, 306- 31 1 ( 1995); Kuriyan & Cowburn, Curr. Opin. Struct. Biol., 3, 828-837 ( 1993); Cohen et al., Cell, 80, 237-248 ( 1995)]. The term "SHI domain" refers to a family of homologous protein domains that bind ATP and catalyze tyrosine phosphorylation of peptide and protein substrates. The term "SH2 domain" refers to a family of homologous protein domains that share the common property of recognizing phosphorylated tyrosine residues in specific peptide contexts. The term "SH3 domain" refers to a family of homologous protein domains that share the common property of recognizing polyproline type II helices. The term "PH domain" refers to a family of homologous protein domains that mediate both protein-protein and protein-lipid interactions. Examples of SH2 domains which may be utilized in the method of the invention include, but are not limited to, the single and tandem SH2 domains present in the tyrosine kinases ZAP, SYK and LCK. The DNA sequences were obtained from GenBank, National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894. The Accession Numbers for the sequences are: human ZAP (L05148); human SYK (L28824) and human LCK (X I 3529).
The term "tagged ligand" refers to a biotinylated or epitope tagged ligand for the target protein.
The term "radiolabeled ligand" refers to a H]-, [125j]_ [ 14C]., [35s]-, [32p]_, or [33P]_labeled ligand which binds to the FKBP. An example of a radiolabeled ligand useful in the instant invention is [3H]-dihydroFK506.
The term "coated microscintillation plates" refers to streptavidin-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 radiolabeled ligand and the coated microscintillation plates, but in the absence of the test compound.
The term FK506-binding proteins may include, but are not limited to, the below listed FKBPs and FKBP homologues, which include a citation to the references which disclose them. This list is not intended to limit the scope of the invention.
Mammalian
FKBP-12 Galat et al., Eur. J. Biochem., 216:689-
707 (1993). FKBP-12.6 Wiederrecht, G. and F. Etzkorn Perspectives in Drug Discovery and
Design , 2:57-84 (1994). FKBP-13 Galat et al., supra', Wiederrecht and
Etzkorn, supra. FKBP-25 Galat et al., supra; Wiederrecht and Etzkorn, supra.
FKBP-38 Wiederrecht and Etzkorn, supra.
FKBP-51 Baughman et al., Mol. Cell. Biol., 8,
4395-4402(1995) . FKBP-52 Galat et al., supra. Bacteria
Legionella pneumophilia Galat et al., supra. Legionella micadei Galat et al., supra. Chlamydia trachomatis Galat et al., supra. E. coli fkpa Home, S.M. and K.D. Young, Arch.
Microbiol., 163:357-365 (1995).
E. coli slyD Roof et al., J. Biol. Chem. 269:2902-
2910 ( 1994). E. coli orf 149 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.
Funyal yeast FKBP- 12 Cardenas et al., Perspectives in Drug
Discovery and Design , 2: 103-126
(1994). yeast FKBP- 13 Cardenas et al., supra. yeast NPR 1(FPR3) Cardenas et al., supra. Neurospora Galat et al., supra.
A variety of host cells may be used in this invention, which include, but are not limited to, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express genes in a variety of host cells, such as, bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Commercially available vectors suitable for FKBP fusion protein expression include, but are not limited to pBR322 (Promega), pGEX (Amersham), pT7 (USB), pET (Novagen), pIBI (IB I), pProEX-1 (Gibco/BRL), pBluescript II (Stratagene), pTZ18R and pTZ19R (USB), pSE420 (Invitrogen), pVL1392 (Invitrogen), pBlueBac (Invitrogen), pBAcPAK (Clontech), pHIL (Invitrogen), pYES2 (Invitrogen), pCDNA (Invitrogen), pREP (Invitrogen) or the like.
The expression vector may be introduced into host cells via any one of a number of techinques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation.
E. coli containing an expression plasmid with the target gene fused to FKBP are grown and appropriately induced. The cells are then pelleted and resuspended in a suitable buffer. Although FKBP- 12 lacks sequences that specifically direct it to the periplasm, FKBP fusions are primarily located there and can be released by a standard freeze/thaw treatment of the cell pellet. Following centrifugation, the resulting supernatant contains >80% pure FKBP fusion, which if desired can be purified further by conventional methods. Altematively, the assay is not dependent on pure protein and the initial periplasmic preparation may be used directly. A t-hrombin site located between FKBP and the target protein can be used as a means to cleave FKBP from the fusion; such cleaved material may be a suitable negative control for subsequent assays. A fusion protein which contains a single or multiple SH2 domain(s) may be purified by preparing an affinity matrix consisting of biotinylated phosphopeptide coupled to avidin or streptavidin immobilized on a solid support. A freeze/thaw extract is prepared from the cells which express the fusion protein and is loaded onto the affinity matrix. The desired fusion protein is then specifically eluted with phenyl phosphate.
To assay the formation of a complex between a target protein and its ligand, the tagged ligand is mixed with the FKBP fusion protein in a suitable buffer in the presence of the radiolabeled ligand. After a suitable incubation period to allow complex formation to occur, the mixture is transferred to a coated microscintillation plate to capture the tagged ligand and any bound fusion protein. The plate is sealed, incubated for a sufficient period to allow the capture to go to completion, then counted in a multiwell scintillation counter. Screening for agonists/antagonists/inhibitors is carried out by performing the initial incubation prior to the capture step in the microscintillation plate 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 invention.
EXAMPLE 1
Process for Preparing the FKBP fusion cloning vector
General techniques for modifying and expressing genes in various host cells can be found in Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. Current Protocols in Molecular Biology (John Wiley & Sons, New York, New York, 1989). Sequences for a 3'- altered FKBP fragment that contained a glycine codon (GGT) in place of the stop (TGA) codon followed by a sequence encoding a thrombin site (Leu-Val-Pro-Arg) and BamHI restriction site (GAATTC) were amplified using the polymerase chain reaction (PCR). The PCR reaction contained the following primers :5'- GATCGCCATGGGAGTGCAGGTGGAAACCATCTCCCCA-3' and 5 - TACGAATTCTGGCGTGGATCCACGCGGAACCAGACCTTCCAGT TTTAG-3' and a plasmid containing human FKBP-12 as the template. The resulting 367 base pair amplification product was ligated into the vector pCRII (Invitrogen) and the ligation mixture transformed into competent Escherichia coli cells. Clones containing an insert were identified using PCR with flanking vector primers. Dideoxy DNA sequencing confirmed the nucleotide sequence of one positive isolate. The altered 338 base pair FKBP fragment was excised from the pCRII plasmid using Ncol and BamHI and ligated into Ncol and/fømHI digested pET9d (Novagen) plasmid. Competent E. coli were transformed with the ligation mixture, and colonies containing the insert were identified using PCR with primers encoding for flanking vector sequences. The FKBP fusion cloning vector is called pET9dFKBPt.
EXAMPLE 2
Process for Preparing the FK-ZAP fusion expression vector A DNA fragment encoding for the tandem SH2 domains of
ZAP-70 was prepared by PCR to contain a BamHI site at the 5 '-end such that the reading frame was conserved with that of FKBP in the fusion vector. At the 3'-end, the fragment also incoφorated a stop codon followed by a BamHI site. The PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
5 -ATTAGGATCCATGCCAGATCCTGCAGCTCACCTGCCCT-3' and 5 -ATATGGATCCTTACCAGAGGCGTTGCT-3'. The fragment was cloned into a suitable vector, sequenced, digested with BamHI, and the insert containing the SH2 domains ligated to BamHI treated pET9dFKBPt, and transformed into E. coli. Clones containing inserts in the correct orientation were identified by PCR or restriction enzyme analysis. Plasmid DNA was prepared and used to transform BL21(DE3) cells. EXAMPLE 3
Process for Preparing the FK-SYK fusion expression vector
The expression vector for the tandem SH2 domains of Syk fused to FKBP was prepared as in Example 2 except that the PCR reaction contained Raji cell cDNA (Clontech) and the following primers: 5 -CAATAGGATCCATGGCCAGCAGCGGCATGGCTGA-3' and 5 -GACCTAGGATCCCTAATTAACATTTCCCTGTGTGCCGAT- 3* .
EXAMPLE 4
Process for Preparing the FK-LCK fusion expression vector
The expression vector for the SH2 domain of Lck fused to FKBP was prepared as in Example 2 except that the PCR reaction contained Molt-4 cDNA (Clontech) and the following primers:
5 -ATATGGATCCATGGCGAACAGCCTGGAGCCCGAACCCT-3' and 5 -ATTAGGATCCTTAGGTCTGGCAGGGGCGGCTCAACCGTG TGC A-3' .
EXAMPLE 5
FK-ZAP
Step A: Process for Expression of FK-ZAP
E. coli BL21 (DE3) cells containing the pET9dFKBPt/ ZapSH2 plasmid were grown in Luria-Bertani (LB) media containing 50 microgram/ml kanamycin at about 37 degrees C until the optical density measured at 600 nm was about 0.5-1.0. Expression of the FK-ZAP fusion protein was induced with 0.1 mM isopropyl beta- thiogalactopyranoside and the cells were grown for another 3-5 hr at 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, 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 prepared by combining agarose-immobilized avidin with excess biotinylated phosphopeptide derived from the ζl ITAM sequence of the human T-cell receptor, biotinyl-GSNQLpYNELNLGRREEpYDVLDK, and washing out unbound peptide. Frozen cells containing FK-ZAP were thawed in warm water, refrozen on dry ice for about 25 min., then thawed again. After the addition of 0.1 % octyl glucoside, 1 mM dithiothreitol (DTT) and 500 mM NaCl, the extract was centrifuged at 35,000 x g for approximately 30 minutes. The supernatant was loaded onto the phosphopeptide affinity column, at about 4° and washed with phosphate buffered saline containing 1 mM DTT and 0.1 % octyl glucoside. FK-ZAP was eluted with 200 mM phenyl phosphate in the same buffer at about 37°. The protein pool was concentrated and the phenyl phosphate removed on a desalting column. The purified FK- ZAP was stored at about -30° in 10 mM HEPES/150 mM NaCl/1 mM DTT/0.1 mM EDT A/10% glycerol.
EXAMPLE 6 FK-SYK
E. coli BL21 (DE3) cells containing the pET9dFKBPt/
SykSH2 plasmid were grown, induced, and harvested as described in Example 5. FK-SYK was purified using the same affinity matrix and methodology described in Example 5.
EXAMPLE 7
FK-LCK
E. coli BL21 (DE3) cells containing the pET9dFKBPt/ LckSH2 plasmid were grown, induced, and harvested as described in Example 5. The affinity matrix for purification of FK-LCK was prepared by combining agarose-immobilized avidin with excess biotinyl- EPQpYEEIPIYL, and washing out unbound peptide. The remaining methodology for purification was the same as Example 5.
EXAMPLE 8
Assav of phosphopeptide binding to FK-ZAP
Assays were conducted at ambient temperature in a buffer consisting of 25 mM HEPES, 10 mM DTT, 0.01 % TWEEN-20, pH 7.0. 300 μl of a mixture of buffer and varying amounts of biotinyl- phosphopeptide were combined with 25 μl of FK-ZAP protein and 50 μl of [3H]-dihydroFK506 (DuPont NEN) in microfuge tubes. A 150 μl portion of each assay was then transferred to the well of a streptavidin- coated FlashPlate Plus (DuPont-NEN) and an additional 50 μl of buffer was added. Final concentrations of the assay components were:
0-50 nM biotinyl-GSNQLpYNELNLGRREEpYDVLDK 100 nM FK-ZAP fusion protein 25 nM [3H]-dihydroFK506 The plate was sealed and incubated 20 hours. Plate -bound radioactivity was measured at various timepoints in a Packard Topcount microplate scintillation counter.
EXAMPLE 9
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 7.0. 10 μl of a DMSO solution of test compound(s) and 120 μl of biotinyl- phosphopeptide stock solution are dispensed into the wells of a standard 96-well plate. Next, 20 μl 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
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 scintillation counter.
EXAMPLE 10
Method of Screening for Antagonists of FK-SYK
The assays are conducted as set forth in Example 9, except that FK-SYK replaces FK-ZAP.
EXAMPLE 1 1
Method of Screening for Antagonists of FK-LCK
The assays are conducted as set forth in Example 9, except that FK-LCK replaces FK-ZAP and the tagged ligand is 25 nM biotinyl-EPQpYEEIPIYL.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Salowe, Scott P. Marcy, Alice I. Wisniewski, Douglas
(ii) TITLE OF INVENTION: A High Throughput Assay Using Fusion Proteins
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Valerie J. Camara
(B) STREET: 126 E. Lincoln Avenue, P.O. Box 2000
(C) CITY: Rahway
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(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Camara, Valerie J.
(B) REGISTRATION NUMBER: 35,090
(C) REFERENCE/DOCKET NUMBER: 19524
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908) 594-3902
(B) TELEFAX: (908) 594-4720
(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
( i i ) MOLECULE TYPE : DNA ( genomic ) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :
ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60
CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 120
CGGGACAGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 180
GAAGAAGGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240
TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTTC 300
GATGTGGAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGCC AGATCCTGCA 360
GCTCACCTGC CCTTCTTCTA CGGCAGCATC TCGCGTGCCG AGGCCGAGGA GCACCTGAAG 420
CTGGCGGGCA TGGCGGACGG GCTCTTCCTG CTGCGCCAGT GCCTGCGCTC GCTGGGCGGC 480
TATGTGCTGT CGCTCGTGCA CGATGTGCGC TTCCACCACT TTCCCATCGA GCGCCAGCTC 540
AACGGCACCT ACGCCATTGC CGGCGGCAAA GCGCACTGTG GACCGGCAGA GCTCTGCGAG 600
TTCTACTCGC GCGACCCCGA CGGGCTGCCC TGCAACCTGC GCAAGCCGTG CAACCGGCCG 660
TCGGGCCTCG AGCCGCAGCC GGGGGTCTTC GACTGCCTGC GAGACGCCAT GGTGCGTGAC 720
TACGTGCGCC AGACGTGGAA GCTGGAGGGC GAGGCCCTGG AGCAGGCCAT CATCAGCCAG 780
GCCCCGCAGG TGGAGAAGCT CATTGCTACG ACGGCCCACG AGCGGATGCC CTGGTACCAC 840
AGCAGCCTGA CGCGTGAGGA GGCCGAGCGT AAACTTTACT CTGGGGCGCA GACCGACGGC 900
AAGTTCCTGC TGAGGCCGCG GAAGGAGCAG GGCACATACG CCCTGTCCCT CATCTATGGG 960
AAGACGGTGT ACCACTACCT CATCAGCCAA GACAAGGCGG GCAAGTACTG CATTCCCGAG 1020
GGCACCAAGT TTGACACGCT CTGGCAGCTG GTGGAGTATC TGAAGCTGAA GGCGGACGGG 1080
CTCATCTACT GCCTGAAGGA GGCCTGCCCC AACAGCAGTG CCAGCAACGC CTCTTAA 1137 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1155 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :
ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60 CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 120
CGGGACAGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 180
GAAGAAGGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240
TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTTC 300
GATGTGGAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGGC CAGCAGCGGC 360
ATGGCTGACA GCGCCAACCA CCTGCCCTTC TTTTTCGGCA ACATCACCCG GGAGGAGGCA 420
GAAGATTACC TGGTCCAGGG GGGCATGAGT GATGGGCTTT ATTTGCTGCG CCAGAGCCGC 480
AACTACCTGG GTGGCTTCGC CCTGTCCGTG GCCCACGGGA GGAAGGCACA CCACTACACC 540
ATCGAGCGGG AGCTGAATGG CACCTACGCC ATCGCCGGTG GCAGGACCCA TGCCAGCCCC 600
GCCGACCTCT GCCACTACCA CTCCCAGGAG TCTGATGGCC TGGTCTGCCT CCTCAAGAAG 660
CCCTTCAACC GGCCCCAAGG GGTGCAGCCC AAGACTGGGC CCTTTGAGGA TTTGAAGGAA 720
AACCTCATCA GGGAATATGT GAAGCAGACA TGGAACCTGC AGGGTCAGGC TCTGGAGCAG 780
GCCATCATCA GTCAGAAGCC TCAGCTGGAG AAGCTGATCG CTACCACAGC CCATGAAAAA 840
ATGCCTTGGT TCCATGGAAA AATCTCTCGG GAAGAATCTG AGCAAATTGT CCTGATAGGA 900
TCAAAGACAA ATGGAAAGTT CCTGATCCGA GCCAGAGACA ACAACGGCTC CTACGCCCTG 960
TGCCTGCTGC ACGAAGGGAA GGTGCTGCAC TATCGCATCG ACAAAGACAA GACAGGGAAG 1020
CTCTCCATCC CCGAGGGAAA GAAGTTCGAC ACGCTCTGGC AGCTAGTCGA GCATTATTCT 1080
TATAAAGCAG ATGGTTTGTT AAGAGTTCTT ACTGTCCCAT GTCAAAAAAT CGGCACACAG 1140
GGAAATGTTA ATTAG 1155 (2) INFORMATION FOR SEQ ID NO:3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 675 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :
ATGGGAGTGC AGGTGGAAAC CATCTCCCCA GGAGATGGAC GCACCTTCCC CAAGCGCGGC 60 CAGACCTGCG TGGTGCACTA CACCGGGATG CTTGAAGATG GAAAGAAATT TGATTCCTCC 120
CGGGACAGAA ACAAGCCCTT TAAGTTTATG CTAGGCAAGC AGGAGGTGAT CCGAGGCTGG 180
GAAGAAGGGG TTGCCCAGAT GAGTGTGGGT CAGAGAGCCA AACTGACTAT ATCTCCAGAT 240
TATGCCTATG GTGCCACTGG GCACCCAGGC ATCATCCCAC CACATGCCAC TCTCGTCTTC 300
GATGTGGAGC TTCTAAAACT GGAAGGTCTG GTTCCGCGTG GATCCATGGC GAACAGCCTG 360
GAGCCCGAAC CCTGGTTCTT CAAGAACCTG AGCCGCAAGG ACGCGGAGCG GCAGCTCCTG 420
GCGCCCGGGA ACACTCACGG CTCCTTCCTC ATCCGGGAGA GCGAGAGCAC CGCGGGATCG 480
TTTTCACTGT CGGTCCGGGA CTTCGACCAG AACCAGGGAG AGGTGGTGAA ACATTACAAG 540
ATCCGTAATC TGGACAACGG TGGCTTCTAC ATCTCCCCTC GAATCACTTT TCCCGGCCTG 600
CATGAACTGG TCCGCCATTA CACCAATGCT TCAGATGGGC TGTGCACACG GTTGAGCCGC 660
CCCTGCCAGA CCTAA 675 (2) INFORMATION FOR SEQ ID 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 Gin Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15
Pro Lys Arg Gly Gin Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30
Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45
Phe Met Leu Gly Lys Gin Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60
Ala Gin Met Ser Val Gly Gin Arg Ala Lys Leu Thr Ile Ser Pro Asp 65 70 75 80
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro 100 105 110
Arg Gly Ser Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly 115 120 125
Ser Ile Ser Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met 130 135 140
Ala Asp Gly Leu Phe Leu Leu Arg Gin Cys Leu Arg Ser Leu Gly Gly 145 150 155 160
Tyr Val Leu Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile 165 170 175
Glu Arg Gin Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His 180 185 190
Cys Gly Pro Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly 195 200 205
Leu Pro Cys Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu 210 215 220
Pro Gin Pro Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp 225 230 235 240
Tyr Val Arg Gin Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gin Ala 245 250 255
Ile Ile Ser Gin Ala Pro Gin Val Glu Lys Leu Ile Ala Thr Thr Ala 260 265 270
His Glu Arg Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala 275 280 285
Glu Arg Lys Leu Tyr Ser Gly Ala Gin Thr Asp Gly Lys Phe Leu Leu 290 295 300
Arg Pro Arg Lys Glu Gin Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly 305 310 315 320
Lys Thr Val Tyr His Tyr Leu Ile Ser Gin Asp Lys Ala Gly Lys Tyr 325 330 335
Cys Ile Pro Glu Gly Thr Lys Phe Asp Thr Leu Trp Gin Leu Val Glu 340 345 350
Tyr Leu Lys Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala 355 360 365
Cys Pro Asn Ser Ser Ala Ser Asn Ala Ser 370 375
(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 Gin Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15
Pro Lys Arg Gly Gin Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30
Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45
Phe Met Leu Gly Lys Gin Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60
Ala Gin Met Ser Val Gly Gin Arg Ala Lys Leu Thr Ile Ser Pro Asp 65 70 75 80
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly lie Ile Pro Pro His Ala 85 90 95
Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro 100 105 110
Arg Gly Ser Met Ala Ser Ser Gly Met Ala Asp Ser Ala Asn His Leu 115 120 125
Pro Phe Phe Phe Gly Asn Ile Thr Arg Glu Glu Ala Glu Asp Tyr Leu 130 135 140
Val Gin Gly Gly Met Ser Asp Gly Leu Tyr Leu Leu Arg Gin Ser Arg 145 150 155 160
Asn Tyr Leu Gly Gly Phe Ala Leu Ser Val Ala His Gly Arg Lys Ala 165 170 175
His His Tyr Thr Ile Glu Arg Glu Leu Asn Gly Thr Tyr Ala Ile Ala 180 185 190
Gly Gly Arg Thr His Ala Ser Pro Ala Asp Leu Cys His Tyr His Ser 195 200 205
Gin Glu Ser Asp Gly Leu Val Cys Leu Leu Lys Lys Pro Phe Asn Arg 210 215 220 Pro Gin Gly Val Gin Pro Lys Thr Gly Pro Phe Glu Asp Leu Lys Glu 225 230 235 240
Asn Leu Ile Arg Glu Tyr Val Lys Gin Thr Trp Asn Leu Gin Gly Gin 245 250 255
Ala Leu Glu Gin Ala Ile Ile Ser Gin Lys Pro Gin Leu Glu Lys Leu 260 265 270
Ile Ala Thr Thr Ala His Glu Lys Met Pro Trp Phe His Gly Lys Ile 275 280 285
Ser Arg Glu Glu Ser Glu Gin Ile Val Leu Ile Gly Ser Lys Thr Asn 290 295 300
Gly Lys Phe Leu Ile Arg Ala Arg Asp Asn Asn Gly Ser Tyr Ala Leu 305 310 315 320
Cys Leu Leu His Glu Gly Lys Val Leu His Tyr Arg Ile Asp Lys Asp 325 330 335
Lys Thr Gly Lys Leu Ser Ile Pro Glu Gly Lys Lys Phe Asp Thr Leu 340 345 350
Trp Gin Leu Val Glu His Tyr Ser Tyr Lys Ala Asp Gly Leu Leu Arg 355 360 365
Val Leu Thr Val Pro Cys Gin Lys Ile Gly Thr Gin Gly Asn Val Asn 370 375 380
(2) INFORMATION FOR SEQ ID NO: 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Gly Val Gin Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15
Pro Lys Arg Gly Gin Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30
Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45 Phe Met Leu Gly Lys Gin Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60
Ala Gin Met Ser Val Gly Gin Arg Ala Lys Leu Thr Ile Ser Pro Asp
65 70 75 80
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95
Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Leu Val Pro 100 105 110
Arg Gly Ser Met Ala Asn Ser Leu Glu Pro Glu Pro Trp Phe Phe Lys 115 120 125
Asn Leu Ser Arg Lys Asp Ala Glu Arg Gin Leu Leu Ala Pro Gly Asn 130 135 140
Thr His Gly Ser Phe Leu Ile Arg Glu Ser Glu Ser Thr Ala Gly Ser 145 150 155 160
Phe Ser Leu Ser Val Arg Asp Phe Asp Gin Asn Gin Gly Glu Val Val 165 170 175
Lys His Tyr Lys Ile Arg Asn Leu Asp Asn Gly Gly Phe Tyr Ile Ser 180 185 190
Pro Arg Ile Thr Phe Pro Gly Leu His Glu Leu Val Arg His Tyr Thr 195 200 205
Asn Ala Ser Asp Gly Leu Cys Thr Arg Leu Ser Arg Pro Cys Gin Thr 210 215 220

Claims

WHAT IS CLAIMED IS:
1. A method of screening for compounds capable of binding to a fusion protein which comprises the steps of: a) mixing a test compound, a tagged ligand, the fusion protein, 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]-, [125l]., [ 14C]-, [35S]., [32p]_, or [33p]-iabeled 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: SHI , SH2, SH3 and PH domains.
8. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 7, wherein the target protein is a single or multiple SH2 domain.
9. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 8, wherein the radiolabeled ligand is [3H] -dihydro FK506.
10. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 9, wherein the FK506- binding protein is a 12kDA human FK506-binding protein.
1 1. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 10, wherein the target protein is a single or multiple SH2 domain selected from the group consisting of: ZAP:SH2, SYK:SH2 and LCK:SH2.
12. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 1 1 , wherein the target protein is the SH2 domain, ZAP:SH2.
13. The method of screening for compounds capable of binding to a fusion protein, as recited in Claim 1 1 , 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 1 1 , 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.
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.
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: SHI , 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.
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.
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.
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.
22. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in Claim 21 , wherein the expression vector is selected from the group consisting of: pBR322, pGEX, pT7, pET, pIBI, pProEX-1 , pBluescript II, pTZ18R and pTZ19R, pSE420, pVL1392, pBlueBac, pBAcPAK, pHIL, pYES2, pCDNA, and pREP.
23. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in Claim 22, wherein the expression vector is the T7 RNA polymerase based pET expression vector.
24. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in Claim 23, wherein the target protein is ZAP:SH2.
25. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in Claim 23, wherein the target protein is SYK:SH2.
26. The process for preparing a recombinant DNA expression vector encoding the fusion protein, as recited in Claim 23, wherein the target protein is LCK:SH2.
27. Isolated DNA encoding for a fusion protein comprising the sequence: (SEQ. ID. NO. 1).
28. Isolated DNA encoding for a fusion protein comprising the sequence:
(SEQ. ID. NO. 2).
29. Isolated DNA encoding for a fusion protein comprising the sequence: (SEQ. ID. NO. 3).
30. A FKBP-ZAP:SH2 fusion protein comprising the sequence:
(SEQ. ID. NO. 4).
31. A FKBP-S YK :SH2 fusion protein comprising the sequence:
(SEQ. ID. NO. 5).
32. A FKBP-LCK:SH2 fusion protein comprising the sequence: (SEQ. ID. NO. 6).
33. 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.
34. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 33, wherein the target protein is a single or multiple signal transduction domain.
35. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 34, wherein the single or multiple signal transduction domain is selected from the group consisting of: SHI , SH2, SH3 and PH domains.
36. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 35, wherein the single or multiple signal transduction domain is a single or multiple SH2 domain.
37. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 36, wherein the single or multiple SH2 domain is selected from a group consisting of ZAP:SH2, SYK:SH2 and LCK:SH2.
38. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 37, wherein the FK506- binding protein is human 12kDa FK506-binding protein.
39. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 38, wherein the host cell is from bacteria, yeast, blue green algae, plant cells, insect cells, or animal cells.
40. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 39, wherein the expression vector is T7 RNA polymerase based expression vector.
41. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 40, wherein the host cell is an E. coli strain selected from a group consisting of BL21 (DE3), Nova Blue (DE3), and JM109 (DE3).
42. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 41 , wherein the single or multiple SH2 domain is ZAP:SH2.
43. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 41 , wherein the single or multiple SH2 domain is SYK:SH2.
44. The process for expressing recombinant DNA encoding a fusion protein, as recited in Claim 41 , wherein the single or multiple SH2 domain is LCK:SH2.
45. 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.
46. 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).
47. 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).
48. 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).
49. 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).
50. The recombinant host cell containing the recombinant FKBP-SH2 domain T7 RNA polymerase-based expression vector as recited in claim 49, wherein the recombinant host cell is E. coli BL21 (DE3).
PCT/US1996/014567 1995-09-15 1996-09-11 A high throughput assay using fusion proteins WO1997010253A1 (en)

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