DE69941946D1 - EIWEISBRUCHSTÜCKERGÄNZUNGSTESTS - Google Patents

EIWEISBRUCHSTÜCKERGÄNZUNGSTESTS

Info

Publication number
DE69941946D1
DE69941946D1 DE69941946T DE69941946T DE69941946D1 DE 69941946 D1 DE69941946 D1 DE 69941946D1 DE 69941946 T DE69941946 T DE 69941946T DE 69941946 T DE69941946 T DE 69941946T DE 69941946 D1 DE69941946 D1 DE 69941946D1
Authority
DE
Germany
Prior art keywords
protein
dhfr
fragment
fragments
interactions
Prior art date
Legal status (The legal status 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 status listed.)
Expired - Lifetime
Application number
DE69941946T
Other languages
German (de)
Inventor
Stephen William Michnick
Joelle Nina Pelletier
Ingrid Remy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Odyssey Pharmaceuticals Inc
Original Assignee
Odyssey Pharmaceuticals 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 US09/124,850 external-priority patent/US6294330B1/en
Application filed by Odyssey Pharmaceuticals Inc filed Critical Odyssey Pharmaceuticals Inc
Priority claimed from PCT/CA1999/000702 external-priority patent/WO2000007038A2/en
Application granted granted Critical
Publication of DE69941946D1 publication Critical patent/DE69941946D1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Abstract

We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. A Protein Complementation Assay/Universal Reporter System (PCA/URS) for detecting and screening for agonists and antagonists of a membrane receptor is also described. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and Cterminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E. coli doubling times illustrating the successful DHFR fragment reassembly rather than non-specific interactions between fragments. This assay could be used to study equilibrium and kinetic aspects of molecular interactions including protein-protein, protein-DNA, protein-RNA, protein-carbohydrate and protein-small molecule interactions, for screening cDNA libraries for binding of a target protein with unknown proteins or libraries of small organic molecules for biological activity. The selection and design criteria applied here is developed for numerous examples of clonal selection, colorometric, fluorometric and other assays based on enzymes whose products can be measured. The development of such assay systems is shown to be simple, and provides for a diverse set of protein fragment complementation applications.
DE69941946T 1998-07-30 1999-07-30 EIWEISBRUCHSTÜCKERGÄNZUNGSTESTS Expired - Lifetime DE69941946D1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CA002244349A CA2244349A1 (en) 1998-07-30 1998-07-30 Protein fragment complementation assays for the detection of biological or drug interactions
US09/124,850 US6294330B1 (en) 1997-01-31 1998-07-30 Protein fragment complementation assays for the detection of biological or drug interactions
PCT/CA1999/000702 WO2000007038A2 (en) 1998-07-30 1999-07-30 Protein fragment complementation assays

Publications (1)

Publication Number Publication Date
DE69941946D1 true DE69941946D1 (en) 2010-03-11

Family

ID=29409708

Family Applications (1)

Application Number Title Priority Date Filing Date
DE69941946T Expired - Lifetime DE69941946D1 (en) 1998-07-30 1999-07-30 EIWEISBRUCHSTÜCKERGÄNZUNGSTESTS

Country Status (5)

Country Link
AT (1) ATE456054T1 (en)
CA (1) CA2244349A1 (en)
DE (1) DE69941946D1 (en)
DK (1) DK1027608T3 (en)
ES (1) ES2339411T3 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7488583B2 (en) * 2003-09-25 2009-02-10 Odyssey Thera, Inc. Fragment complementation assays for G-protein-coupled receptors and their signaling pathways
CN114190538B (en) * 2021-12-21 2023-11-24 郑州轻工业大学 Grease crystallization promoter for promoting formation of beta' crystal form and application

Also Published As

Publication number Publication date
ES2339411T3 (en) 2010-05-19
CA2244349A1 (en) 2000-01-30
ATE456054T1 (en) 2010-02-15
DK1027608T3 (en) 2010-05-25

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