CA2279329C - Analyses par complementation de fragments proteiques pour detecter des interactions biomoleculaires - Google Patents

Analyses par complementation de fragments proteiques pour detecter des interactions biomoleculaires Download PDF

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Publication number
CA2279329C
CA2279329C CA2279329A CA2279329A CA2279329C CA 2279329 C CA2279329 C CA 2279329C CA 2279329 A CA2279329 A CA 2279329A CA 2279329 A CA2279329 A CA 2279329A CA 2279329 C CA2279329 C CA 2279329C
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Prior art keywords
protein
fragments
molecule
fragment
assay
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Expired - Fee Related
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CA2279329A
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CA2279329A1 (fr
Inventor
Stephen William Watson Michnick
Joelle Nina Pelletier
Ingrid Remy
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Odyssey Pharmaceuticals Inc
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Odyssey Pharmaceuticals Inc
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Priority claimed from CA002196496A external-priority patent/CA2196496A1/fr
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Priority to CA2279329A priority Critical patent/CA2279329C/fr
Publication of CA2279329A1 publication Critical patent/CA2279329A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Nous décrivons une stratégie permettant de créer et de mettre en oeuvre des analyses par complémentation de fragments protéiques (PCA) pour détecter des interactions biomoléculaires in vivo et in vitro. La création, la mise en oeuvre et les larges applications de cette stratégie sont illustrées par un grand nombre d'enzymes, notamment par l'exemple détaillé de l'hydrofolate réductase murine (DHFR). Les peptides de fusion comprenant les fragments N et C-terminaux de la DHFR murine condensés avec les séquences formant des glissières à leucine GCN4 ont été co-exprimées chez Escherichia coli cultivée dans un milieu minimum, chez laquelle l'activité DHFR endogène a été inhibée par le triméthoprime. La co-expression des produits de fusion complémentaires a permis de nouveau la formation de colonies. La survie n'était possible que quand les fragments de DHFR étaient présents et contenaient des séquences formatrices de glissières à leucine, ce qui montre que la formation de ces dernières est nécessaire à la reconstitution de l'activité enzymatique. Les mutations ponctuelles fragment DHFR-interface d'une importance croissante (Ile vers Val, Ala et Gly) ont abouti à un augmentation des temps de doublement d'E. coli, ce qui illustre la réussite du réassemblage des fragments de DHFR plutôt que des interactions non spécifiques entre fragments. Cette analyse a été utilisée pour étudier l'équilibre et les aspects cinétiques des interactions moléculaires, notamment des interactions protéine-protéine, protéine-ADN, protéine-ARN, protéine-glucide et protéine-petite molécule, dans le but de cribler des librairies d'ADNc permettant de lier une protéine cible à des protéines inconnues ou des librairies de petites molécules organiques en vue d'étudier leur activité biologique. Les critères de sélection et de création appliqués ici ont été développés pour de nombreux exemples d'analyses par sélection clonale, d'analyses colorimétriques, fluorimétiques et autres, basées sur des enzymes dont les produits peuvent être dosés. La création de ces systèmes d'analyse, qui s'est avérée simple, permet diverses applications de la complémentation de fragments protéiques.
CA2279329A 1997-01-31 1998-02-02 Analyses par complementation de fragments proteiques pour detecter des interactions biomoleculaires Expired - Fee Related CA2279329C (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA2279329A CA2279329C (fr) 1997-01-31 1998-02-02 Analyses par complementation de fragments proteiques pour detecter des interactions biomoleculaires

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CA2,196,496 1997-01-31
CA002196496A CA2196496A1 (fr) 1997-01-31 1997-01-31 Epreuve de complementation de fragments de proteines pour la detection d'interactions entre proteines
PCT/CA1998/000068 WO1998034120A1 (fr) 1997-01-31 1998-02-02 Analyses par complementation de fragments proteiques pour detecter des interactions biomoleculaires
CA2279329A CA2279329C (fr) 1997-01-31 1998-02-02 Analyses par complementation de fragments proteiques pour detecter des interactions biomoleculaires

Publications (2)

Publication Number Publication Date
CA2279329A1 CA2279329A1 (fr) 1998-08-06
CA2279329C true CA2279329C (fr) 2013-05-21

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Family Applications (1)

Application Number Title Priority Date Filing Date
CA2279329A Expired - Fee Related CA2279329C (fr) 1997-01-31 1998-02-02 Analyses par complementation de fragments proteiques pour detecter des interactions biomoleculaires

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201409558D0 (en) * 2014-05-29 2014-07-16 Ucb Biopharma Sprl Method

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Publication number Publication date
CA2279329A1 (fr) 1998-08-06

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