Abstract
BackgroundHuman interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics.ResultsSplit Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degreeC up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system.ConclusionFluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro, which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu.
Highlights
Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs)
While PPI is conveniently assayed by Protein-fragment complementation assay (PCA) in vivo and in cultured cells, it is often desirable to be performed in vitro, to know whether the interaction is direct or not, since many PPI mediators, inhibitors and enhancers may exist in the complex cellular milieu
FKBP12 is a 12 kD domain of FK506 binding protein (FKBP), which is able to associate with FKBP-rapamycin -associated protein (FRB) depending on an antibiotic, rapamycin (PPI mediator in Figure 1) [12,13]
Summary
Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to -background (S/B) ratio, and reversible response. Porter et al successfully utilized a cell-free transcription/translation system for performing firefly luciferase (Fluc)-based PCA in vitro [11]. Their probes can be prepared in shorter time period than the probes expressed in E. coli, yeast or mammalian cells. We report a PCA using purified interacting proteins fused with split Fluc proteins, to detect PPI in a defined solution. To the best of our knowledge, this will be the first investigation of pure in vitro PPI, based on Fluc PCA
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