A Novel Split Reporter Uncages New Possibilities

Abstract

S plit reporters have emerged as valuable tools for the study and manipulation of biological systems. The development of split reporters takes advantage of the properties of some proteins to become fully functional following the recombination of two or more protein fragments that have no activity on their own. In this issue of ACS Central Science, Jones et al. 1 add a versatile new tool to this expanding toolbox by developing a new split esterase reporter.The most common use of split reporters is for the detection and study of protein−protein interactions (PPIs). Protein-fragment complementation assays (PCAs) use the split reporters to inform on the interaction/proximity between two proteins. In these assays, proteins of interest are covalently linked to fragments of a split reporter. PPI induces recombination of the reporter and generation of a measurable signal (Figure 1A).The last 20 years have seen the development of multiple split proteins with different outputs. Genetic changes, cell survival, enzymatic activities, and more can be used to detect and screen for PPI. Yeast two-hybrid screening, the most widely used PPI screening method, uses a split-transcription factor as a reporter and detects the transcription of a specific gene. Bimolecular fluorescence or luminescence complementation assays (BiFCs and BiLCs, respectively) rely on the recombination of a fluorescent protein 2 or luciferase and its derivatives 3 upon interaction between two proteins of interest. A variety of enzymes have been used as scaffolds for split reporters, providing a variety of read-outs, ranging from colorimetric tests as in the case of the split-β-galactosidase to cell viability (split-β-lactamase). Some split reporters are even compatible with molecular imaging techniques such as positron emission tomography, 4 allowing detection of molecular events at the scale of the whole organism.The newly developed split esterase reporter has the ability to provide multiple read-outs on its own. 1 Indeed, upon A new strategy for proximity-dependent unmasking of small molecules using an engineered enzyme biosensor system.