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WO1997010253A1 - Dosage a rendement eleve utilisant des proteines hybrides - Google Patents

Dosage a rendement eleve utilisant des proteines hybrides 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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WO
WIPO (PCT)
Prior art keywords
fusion protein
recited
protein
expression vector
binding
Prior art date
Application number
PCT/US1996/014567
Other languages
English (en)
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.)
Filing date
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/ja
Priority to EP96935808A priority patent/EP0871648A1/fr
Publication of WO1997010253A1 publication Critical patent/WO1997010253A1/fr

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Classifications

    • 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

L'invention porte sur un dosage à rendement élevé utilisé pour sélectionner des composés capables de se lier à une protéine hybride composée d'une protéine-cible et d'une protéine fixant FK506. L'invention concerne également le procédé de préparation de l'ADN codant la protéine hybride, et d'expression de cet ADN. L'invention se rapporte également aux séquences protéiques et d'ADN de recombinaison pour plusieurs protéines hybrides.
PCT/US1996/014567 1995-09-15 1996-09-11 Dosage a rendement eleve utilisant des proteines hybrides WO1997010253A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP9512071A JPH11513246A (ja) 1995-09-15 1996-09-11 融合タンパク質を用いるハイスループットアッセイ
EP96935808A EP0871648A1 (fr) 1995-09-15 1996-09-11 Dosage a rendement eleve utilisant des proteines hybrides

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US381995P 1995-09-15 1995-09-15
US60/003,819 1995-09-15
GBGB9605210.5A GB9605210D0 (en) 1996-03-12 1996-03-12 A high throughput assay using fusion proteins
GB9605210.5 1996-03-12

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WO1997010253A1 true WO1997010253A1 (fr) 1997-03-20

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PCT/US1996/014567 WO1997010253A1 (fr) 1995-09-15 1996-09-11 Dosage a rendement eleve utilisant des proteines hybrides

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EP (1) EP0871648A1 (fr)
JP (1) JPH11513246A (fr)
CA (1) CA2231330A1 (fr)
WO (1) WO1997010253A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039326A3 (fr) * 1996-04-18 1997-12-24 Ariad Pharma Inc Dosage in vitro a polarisation de fluorescence
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
EP1516928A4 (fr) * 2002-06-25 2006-11-08 Sekisui Chemical Co Ltd Vecteur d'expression, hote, proteine fusionnee, procede pour produire une proteine fusionnee et procede pour produire une proteine
US7947494B2 (en) 2006-01-03 2011-05-24 Roche Diagnostics Operations, Inc. Fusion protein comprising an E. coli chaperone protein and a human chaperone protein
WO2021080427A1 (fr) * 2019-10-23 2021-04-29 Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis Polypeptide chimère pour la régulation de cellules immunitaires

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5352660A (en) * 1991-10-31 1994-10-04 Mount Sinai Hospital Corporation Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system
US5434064A (en) * 1991-01-18 1995-07-18 New York University Expression-cloning method for identifying target proteins for eukaryotic tyrosine kinases and novel target proteins
US5464745A (en) * 1993-03-31 1995-11-07 Novagen, Inc. Protein ligand binding region mapping system
US5498597A (en) * 1992-01-17 1996-03-12 Dana-Farber Cancer Institute, Inc. FKBP-13, an FK506-binding immunophilin
US5534424A (en) * 1992-05-12 1996-07-09 Cemu Bioteknik Ab Chemical method for the analysis of DNA sequences
US5580979A (en) * 1994-03-15 1996-12-03 Trustees Of Tufts University Phosphotyrosine peptidomimetics for inhibiting SH2 domain interactions

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5434064A (en) * 1991-01-18 1995-07-18 New York University Expression-cloning method for identifying target proteins for eukaryotic tyrosine kinases and novel target proteins
US5352660A (en) * 1991-10-31 1994-10-04 Mount Sinai Hospital Corporation Method for assaying for a substance that affects a SH2-phosphorylated ligand regulatory system
US5498597A (en) * 1992-01-17 1996-03-12 Dana-Farber Cancer Institute, Inc. FKBP-13, an FK506-binding immunophilin
US5534424A (en) * 1992-05-12 1996-07-09 Cemu Bioteknik Ab Chemical method for the analysis of DNA sequences
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

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, 05 September 1996, Vol. 240, No. 2, SONATORE et al., "The Utility of FK506-Binding Protein as a Fusion Partner in Scintillation Proximity Assays: Application to SH2 Domains", pages 289-297. *
METHODS IN ENZYMOLOGY, 1990, Vol. 185, STUDIER et al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes", pages 60-89. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 12 July 1996, Vol. 271, No. 28, MULLER et al., "Rapid Identification of Phosphopeptide Ligands for SH2 Domains: Screening of Peptide Libraries by Fluorescence-Activated Bead Sorting", pages 16500-16505. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 16 December 1994, Vol. 269, No. 50, GILMER et al., "Peptide Inhibitors of Src SH3-SH2-Phosphoprotein Interactions", pages 31711-31719. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039326A3 (fr) * 1996-04-18 1997-12-24 Ariad Pharma Inc Dosage in vitro a polarisation de fluorescence
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
US7244575B2 (en) 2001-06-22 2007-07-17 Roche Diagnostics Corporation Soluble complex comprising a retroviral surface glycoprotein
EP1516928A4 (fr) * 2002-06-25 2006-11-08 Sekisui Chemical Co Ltd Vecteur d'expression, hote, proteine fusionnee, procede pour produire une proteine fusionnee et procede pour produire une proteine
US7947494B2 (en) 2006-01-03 2011-05-24 Roche Diagnostics Operations, Inc. Fusion protein comprising an E. coli chaperone protein and a human chaperone protein
US8247206B2 (en) 2006-01-03 2012-08-21 Roche Diagnostics Operations, Inc. Fusion protein comprising an Escherichia coli chaperone protein and a human chaperone protein
WO2021080427A1 (fr) * 2019-10-23 2021-04-29 Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis Polypeptide chimère pour la régulation de cellules immunitaires
CN114651061A (zh) * 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 调节免疫细胞的嵌合多肽
EP4458847A3 (fr) * 2019-10-23 2025-02-26 Stichting Het Nederlands Kanker Instituut- Antoni van Leeuwenhoek Ziekenhuis Polypeptide chimérique pour la régulation de cellules immunitaires
AU2020371039B2 (en) * 2019-10-23 2025-04-10 Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis Chimeric polypeptide for regulating immune cells

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