C - Chemistry – Metallurgy – 12 – Q
Patent
C - Chemistry, Metallurgy
12
Q
C12Q 1/68 (2006.01) C12N 15/10 (2006.01) C12Q 1/25 (2006.01) C12Q 1/26 (2006.01) G01N 33/53 (2006.01) G01N 33/566 (2006.01) G01N 33/58 (2006.01) G01N 33/68 (2006.01)
Patent
CA 2244349
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 C- terminal 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.
Michnick Stephen William Watson
Pelletier Joelle Nina
Remy Ingrid
Goudreau Gage Dubuc
Michnick Stephen William Watson
Pelletier Joelle Nina
Remy Ingrid
Universite de Montreal
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