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

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, beta-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.

Design, selection, and characterization of a split chorismate mutase

“…33 virtually any noncovalent interaction amenable to screening. 39 On the other hand, in vivo selection experiments enable the simultaneous interrogation of much larger libraries of potential protein-protein interaction partners. The CM selection system is particularly well characterized, making the split CM a potentially useful addition to the toolkit of split sensors.…”

From jellyfish to biosensors: the use of fluorescent proteins in plants

“…(Magliery et al, 2005;Nyfeler et al, 2005). Unlike FP-based approaches, split-luciferase reporters are reversible (Porter et al, 2008) and have been successfully adapted to establish membrane protein topology (Zamyatnin et al, 2006).…”

A Comprehensive Panel of Turn‐On Caspase Biosensors for Investigating Caspase Specificity and Caspase Activation Pathways

“…Caspase 10 exhibited a specificity profile very similar to caspase 8, however, unlike caspase 8, also displayed activity against WEHD and VDVAD substrates and was clearly the least specific of all the tested caspases. In order to further investigate caspase biosensor specificity as a function of sensor geometry, we also compared the cyclically permuted firefly luciferase DEVD (caspase 3/7) sensor recently reported by Wood and co-workers, [37] with our caspase 3/7 sensor. Both sensors exhibited similar signal-to-background ratios, upon addition of caspase 7, under our experimental conditions ( Figure S6 A in the Supporting Information).…”

“…[36] This class of designed biosensors utilizes either split-firefly luciferase [37] or split-b-lactamase [38] appended to designed coiled coils, in which complementation of the split-protein domains is autoinhibited in the absence of the protease of interest (Figure 1 A). However, upon addition of a specific protease, autoinhibition is released and the split-signaling domains can reassemble and restore luciferase activity.…”