MICHNICK, STEPHEN WILLIAM WATSON,PELLETIER, JOELLE NINA,REMY, INGRID
申请号：
CA2279329
公开号：
CA2279329C
申请日:
1998.02.02
申请国别(地区):
CA
年份:
2013
代理人:
摘要：
We describe a strategy for designing and implementing protein-fragmentcomplementation assays (PCAs) to detect biomolecular interactions in vivo andin vitro. The design, implementation and broad applications of this strategyare illustrated with a large number of enzymes with particular detail providedfor the example of murine dihydrofolate reductase (DHFR). Fusion peptidesconsisting of N and C-terminal fragments of murine DHFR fused to GCN4 leucinezipper 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 containedleucine-zipper forming sequences, demonstrating that reconstitution of enzymeactivity 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 thesuccessful DHFR fragment reassembly rather that non-specific interactionsbetween fragments. This assay could be used to study equilibrium and kineticaspects of molecular interactions including protein-protein, protein-DNA,protein-RNA, protein-carbohydrate and protein-small molecule interactions, forscreening cDNA libraries for binding of a target protein with unknown proteinsor libraries of small organic molecules for biological activity. The selectionand design criteria applied here is developed for numerous examples of clonalselection, colorometric, fluorometric and other assays based on enzymes whoseproducts can be measured. The development of such assay systems is shown to besimple, and provides for a diverse set of protein fragment complementationapplications.
