Compositions for protein fragment complementation assays to detect biomolecular interactions

摘要

Compositions for protein fragment complementation assays (PCAs) to detect biomolecular interactions are described and their broad applications illustrated with a large number of enzymes, in particular murine dihydrofolate reductase (DHFR). Fusion peptides consisting of <Italic>N</Italic>- and <Italic>C</Italic>-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in <Italic>E. coli</Italic> with endogenous DHFR activity inhibited by 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 mutants of increasing severity resulted in a sequential increase in <Italic>E-coli</Italic> doubling times illustrating successful DHFR fragment reassembly rather than non-specific interactions between fragments. The selection and design criteria are developed for numerous examples of clonal selection, colorimetric, fluorometric and other assays based on enzymes whose products can be measured.