Abstract:

Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, β-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12−48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein−protein, protein−DNA, and protein−RNA interactions in homogeneous assays with very high sensitivity (22−1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein−nucleic acid interactions and in evaluating antagonists of protein−protein and protein−peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein−protein and protein−nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.