| 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 N - and C -terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in E. coli 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 E-coli 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. |
