A General and Rapid Cell-Free Approach for the Interrogation of Protein−Protein, Protein−DNA, and Protein−RNA Interactions and their Antagonists Utilizing Split-Protein Reporters

摘要: Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest targets chemical intervention. Herein we describe systematic evaluation split-proteins, specifically the green fluorescent protein, β-lactamase, and several luciferases, their ability to function in completely cell-free systems allow extremely rapid sensitive determination wide range without requirement laborious transfection, cell culture, or protein purification (12−48 h). We demonstrate that split-luciferase system particular is amenable directly interrogating protein−protein, protein−DNA, protein−RNA homogeneous assays with very high sensitivity (22−1800 fold) starting from corresponding mRNA DNA. Importantly, show allows (2 h) identification target-site specificity protein−nucleic acid interact...