Detecting protein-protein interactions using Renilla luciferase fusion proteins.

摘要: We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two fusions, soluble reporter fusion and immobilizing target protein. The is an N-terminal Renilla that exhibits high activity. Crude cleared lysates from transfected Cos1 cells express can be used in binding assays with immobilized proteins. Following incubation washing, target-bound proteins produce light coelenterazine substrate, indicating interaction between of interest. As proof principle, we reproduced known, transient interactions Cdc42 GTPase its effector produced were tested recombinant GST-N-WASP CEP5 Using this assay, could detect specific these approximately 50 min. specificity was demonstrated by showing they GTPase-specific GTP-dependent not seen other unrelated These results suggest LAPD method, which both rapid sensitive, may research practical applications.